Microscopy shows that individual sites of DNA replication and transcription of mammalian nuclei segregate into sets of roughly 22 and 16 higher order domains, respectively. Each domain set displayed a distinct network-like appearance, including regions of individual domains and interdigitation of domains between the two networks. These data support a dynamic mosaic model for the higher order arrangement of genomic function inside the cell nuclei.

Microtubule flux in spindles of insect spermatocytes, long-used models for studies on chromosome behavior during meiosis, was revealed after iontophoretic microinjection of rhodamine-conjugated (rh)-tubulin and fluorescent speckle microscopy. In time-lapse movies of crane-fly spermtocytes, fluorescent speckles generated when rh-tubulin incorporated at microtubule plus ends moved poleward through each half-spindle and then were lost from microtubule minus ends at the spindle poles. The average poleward velocity of approximately 0.7 microm/min for speckles within kinetochore microtubules at metaphase increased during anaphase to approximately 0.9 microm/min. Segregating half-bivalents had an average poleward velocity of approximately 0.5 microm/min, about half that of speckles within shortening kinetochore fibers. When injected during anaphase, rhtubulin was incorporated at kinetochores, and kinetochore fiber fluorescence spread poleward as anaphase progressed. The results show that tubulin subunits are added to the plus end of kinetochore microtubules and are removed from their minus ends at the poles, all while attached chromosomes move poleward during anaphase A. The results cannot be explained by a Pac-man model, in which 1) kinetochore-based, minus end-directed motors generate poleward forces for anaphase A and 2) kinetochore microtubules shorten at their plus ends. Rather, in these cells, kinetochore fiber shortening during anaphase A occurs exclusively at the minus ends of kinetochore microtubules.

DNA replication sites (RS) in synchronized HeLa cells have been studied at the electron microscopic level. Using an improved method for detection following the in vivo incorporation of biotin-16-deoxyuridine triphosphate, discrete RS, or foci are observed throughout the S-phase. In particular, the much larger RS or foci typically observed by fluorescence microscopic approaches in mid- and late-S-phase, are found to be composed of smaller discrete foci that are virtually identical in size to the RS observed in early-S-phase. Pulse-chase experiments demonstrate that the RS of early-S-phase are maintained when chased through S-phase and into the next cell generation. Stereologic analysis demonstrates that the relative number of smaller sized foci present at a given time remains constant from early through mid-S-phase with only a slight decrease in late-S-phase. 3-D reconstruction of serial sections reveals a network-like organization of the RS in early-S-phase and confirms that numerous smaller-sized replication foci comprise the larger RS characteristic of late-S-phase.

We have examined the targeting of proliferating cell nuclear antigen (PCNA), an integral component of the mammalian replicative enzyme DNA polymerase delta, with sites of DNA replication by using confocal microscopy and computer image analysis. Labeling (5 min pulse) of DNA replication sites in normal human diploid fibroblast cells (NHF1) with BrdU was followed by immunostaining with PCNA antibodies. A striking degree of colocalization was seen between PCNA and the characteristic patterns of DNA replication sites of early, middle and late S-phase (Nakayasu and Berezney [1989] J. Cell. Biol. 108:1-11). These observations were confirmed by quantitative computer image analysis which revealed that approximately 90% of the PCNA-stained area overlapped with DNA replication sites in early S-phase. Pulse-chase experiments, involving in vivo labeling for replication followed by PCNA staining at later time points, suggested that PCNA disassembles from previously replicated sites and targets to newly active sites of DNA replication. To further study this phenomenon in living cells, stable GFP-PCNA transfectants under the control of a tetracycline-inducible promoter were created in mouse 3T6 cells. Like the endogenous PCNA, GFP-PCNA targeted to sites of replication (approximately 80% colocalization) and demonstrated similar dynamic changes following pulse-chase experiments in fixed cells. Studies of living cells revealed progressive changes in the GFP-PCNA distribution that mimic the replication patterns observed in fixed cells. We conclude that GFP-PCNA targets to DNA replication sites in living cells and is an effective marker for tracking the spatio-temporal dynamics of DNA replication as cells transverse the S-phase.

C 4 photosynthesis typically requires two specialized leaf cell types, bundle sheath (bs) and mesophyll (mp), which provide the foundation for this highly efficient carbon assimilation pathway. In leaves of Flaveria bidentis, a dicotyledonous C 4 plant, ribulose 1,5-bisphosphate carboxylase (rubisco) accumulates only in bs cells surrounding the vascular centers and not in mp cells. This is in contrast to the more common C 3 plants, which accumulate rubisco in all photosynthetic cells. Many previous studies have focused on transcriptional control of C 4 cell type-specificity; however, post-transcriptional regulation has also been implicated in the bs-specific expression of genes encoding the rubisco subunits. In this current study, a biolistic leaf transformation assay has provided direct evidence that the 5-and 3-untranslated regions (UTRs) of F. bidentis FbRbcS1 mRNA (from a nuclear gene encoding the rubisco small subunit), in themselves, confer strong bs cell-specific expression to gfpA reporter gene transcripts when transcribed from a constitutive CaMV promoter. In transformed leaf regions, strong bs cell-specific GFP expression was accompanied by corresponding bs cellspecific accumulation of the constitutively transcribed FbRbcS1 5-UTR-gfpA-3-UTR mRNAs. Control constructs lacking any RbcS mRNA sequences were expressed in all leaf cell types. These findings demonstrate that characteristic cell type-specific FbRbcS1 expression patterns in C 4 leaves can be established entirely by sequences contained within the transcribed UTRs of FbRbcS1 mRNAs. We conclude that selective transcript stabilization (in bs cells) or degradation (in mp cells) plays a key role in determining bs cell-specific localization of the rubisco enzyme.Plants that utilize the highly efficient C 4 pathway of carbon fixation typically possess a Kranz-type leaf anatomy that consists of two distinct photosynthetic cell types (1-4). Bundle sheath (bs) 3 cells occur as a layer around each leaf vein, while mesophyll (mp) cells occur in one or more layers surrounding the vascular associated rings of bs cells. This specialized leaf anatomy compartmentalizes two sets of photosynthetic reactions that make up the C 4 pathway. In the C 4 dicot Flaveria bidentis, atmospheric CO 2 entering via the leaf stomata is initially incorporated into four carbon acids by phosphoenolpyruvate carboxylase, an enzyme found only in the leaf mp cells. The C 4 acids diffuse from mp cells to bs cells, where they are decarboxylated by a photosynthetic malic enzyme. Within bs chloroplasts, the released CO 2 is incorporated into the Calvin cycle by ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco), the primary enzyme of photosynthetic carbon fixation. The specialized anatomical framework and compartmentalized reactions of the C 4 pathway function as a "CO 2 pump" that concentrates CO 2 in bs cells, where rubisco is specifically localized (1, 2, 5, 6). As a result, the carboxylase activity of this enzyme is increased, while its oxygenase activity, which can decrease phot...