Nucleolar assembly of the rRNA processing machinery in living cells

To understand how nuclear machineries are targeted to accurate locations during nuclear assembly, we investigated the pathway of the ribosomal RNA (rRNA) processing machinery towards ribosomal genes (nucleolar organizer regions [NORs]) at exit of mitosis. To follow in living cells two permanently transfected green fluorescence protein-tagged nucleolar proteins, fibrillarin and Nop52, from metaphase to G1, 4-D time-lapse microscopy was used. In early telophase, fibrillarin is concentrated simultaneously in prenucleolar bodies (PNBs) and NORs, whereas PNB-containing Nop52 forms later. These distinct PNBs assemble at the chromosome surface. Analysis of PNB movement does not reveal the migration of PNBs towards the nucleolus, but rather a directional flow between PNBs and between PNBs and the nucleolus, ensuring progressive delivery of proteins into nucleoli. This delivery appeared organized in morphologically distinct structures visible by electron microscopy, suggesting transfer of large complexes. We propose that the temporal order of PNB assembly and disassembly controls nucleolar delivery of these proteins, and that accumulation of processing complexes in the nucleolus is driven by pre-rRNA concentration. Initial nucleolar formation around competent NORs appears to be followed by regroupment of the NORs into a single nucleolus 1 h later to complete the nucleolar assembly. This demonstrates the formation of one functional domain by cooperative interactions between different chromosome territories.     

Nucleolar ontogenesis in the uterine chick germ correlated with morphogenetic events

Nucleolar development in the cleaving chick germ up to the formation of the primary hypoblast was followed through a series of well-defined uterine and early incubated stages both by light and electron microscopy. Well-established criteria of nucleolar morphology were used for determining the developmental stage of onset of rRNA synthesis. By these criteria rRNA synthesis was first observed at midcleavage in uterine stage VII [1] germs. This could be correlated with the first morphogenetic event—the posterio-anteriorly orientated formation of the area pellucida which results in a bilaterally symmetrical blastoderm.     

Nucleolar size in lymphocytes and haemocytes of different species

The number of nucleoli in a cell and nucleolar area vary according to the cell. We compared nucleoli in mammalian circulating lymphocytes and insect circulating haemocytes. An increased nucleolar coefficient correlated with a lowered nucleoli size. The smaller nucleolar size in mammalian lymphocytes indicates a lower proteosynthetic cellular activity in both mammalian lymphocytes and insect haemocytes. Moreover, in insect haemocytes, the smaller size of the nucleoli may reflect a lowered potential to transform into another cell type.     

Effect of cellulase size reduction on activity and accessibility

Summary Reducing the size of crude cellulase from Trichoderma reesei with various proteases showed that while the -glucosidase activity on CMC generally increased, the extent of hydrolysis of hardwood and pretreated hardwood decreased. The rank in the decrease in hydrolytic activity is the same as the rank in the decrease in enzyme adsorption on the hardwood or pretreated hardwood.     

In situ activity of a dominant Prochlorococcus ecotype (eHL‐II) from rRNA content and cell size

In the open ocean genetically diverse clades of the unicellular cyanobacteria Prochlorococcus are biogeographically structured along environmental gradients, yet little is known about their in situ activity. To address this gap, here we use the numerically dominant Prochlorococcus clade eHL‐II (eMIT9312) as a model organism to develop and apply a method to examine their in situ activity using rRNA content and cell size as metrics of cellular physiology. For two representative isolates (MIT9312 and MIT9215) rRNA cell^?1 increases linearly with specific growth rate but is anticorrelated with cell size indicated by flow cytometrically measured (SSC). Although each strain has a unique relationship between cellular rRNA (or cell size) and growth rate, both strains have the same strong positive correlation between rRNA cell^?1 SSC^?1 and growth rate. We field test this approach and observe distinct patterns of eHL‐II clade specific activity (rRNA cell^?1 SSC^?1) with depth that are consistent with patterns of photosynthetic rates. This molecular technique provides unique insight into the ecology of Prochlorococcus and could potentially be expanded to include other microbes to unravel the ecological and biogeochemical contributions of genetically distinct marine side scatter microbes.     

Development of Candida albicans hyphae in different growth media--variations in growth rates, cell dimensions and timing of morphogenetic events

In six liquid culture media, all of which stimulated Candida albicans to grow in the hyphal form, the rates of hyphal extension and increase in cellular ATP concentration, hyphal diameters, times of evagination of hyphae, times of septum formation and positions of septa in the hyphae appeared to vary independently. There were no discernible associations between properties such as length or volume of hyphal compartments at the time of septation and temporal parameters of hyphal growth. The results suggest that growth environment influences many of the processes contributing to hyphal development, but that these processes are not necessarily interrelated.     

Location of rRNA genes in three inbred strains of rat and suppression of rat rRNA activity in rat-human somatic cell hybrids

Nucleolus organizer regions (NORs) of rat chromosomes were stained by the Ag-AS method. The Ag-NORs were found on chromosomes 3, 11 and 12 in the ACI, Wistar Brown and Wistar Lewis inbred strains of rat. The size of the Ag-NOR on each pair of chromosomes varied from strain to strain. Rat-human somatic hybrid cells that retained human and lost some of the rat chromosomes had no Ag-NOR on rat chromosomes 3, 11 or 12. Since NORs can be Ag-stained only if their 18 + 28S rRNA genes are active, the activity of the rat rRNA genes must have been suppressed in the hybrid cells.     

Nucleolar protein Nop12p participates in synthesis of 25S rRNA in Saccharomyces cerevisiae

A genetic screen for mutations synthetically lethal with temperature sensitive alleles of nop2 led to the identification of the nucleolar proteins Nop12p and Nop13p in Saccharomyces cerevisiae. NOP12 was identified by complementation of a synthetic lethal growth phenotype in strain YKW35, which contains a single nonsense mutation at codon 359 in an allele termed nop12-1. Database mining revealed that Nop12p was similar to a related protein, Nop13p. Nop12p and Nop13p are not essential for growth and each contains a single canonical RNA recognition motif (RRM). Both share sequence similarity with Nsr1p, a previously identified, non-essential, RRM-containing nucleolar protein. Likely orthologs of Nop12p were identified in Drosophila and Schizosaccharomyces pombe. Deletion of NOP12 resulted in a cold sensitive (cs) growth phenotype at 15°C and slow growth at 20 and 25°C. Growth of a nop12Δ strain at 15 and 20°C resulted in impaired synthesis of 25S rRNA, but not 18S rRNA. A nop13 null strain did not produce an observable growth phenotype under the laboratory conditions examined. Epitope-tagged Nop12p, which complements the cs growth phenotype and restores normal 25S rRNA levels, was localized to the nucleolus by immunofluorescence microscopy. Epitope-tagged Nop13p was distributed primarily in the nucleolus, with a lesser portion localizing to the nucleoplasm. Thus, Nop12p is a novel nucleolar protein required for pre-25S rRNA processing and normal rates of cell growth at low temperatures.     

Measuring dimensions: the regulation of size and shape

Over many years evidence has accumulated that plants and animals can regulate growth with reference to overall size rather than cell number. Thus, organs and organisms grow until they reach their characteristic size and shape and then they stop - they can even compensate for experimental manipulations that change, over several fold, cell number or average cell size. If the cell size is altered, the organism responds with a change in cell number and vice versa. We look at the Drosophila wing in more detail: here, both extracellular and intracellular regulators have been identified that link cell growth, division and cell survival to final organ size. We discuss a hypothesis that the local steepness of a morphogen gradient is a measure of length in one axis, a measure that is used to determine whether there will be net growth or not.     

Effect of cell culture on 18S rRNA gene sequences in the cultural course of Taxus chinensis cells

Cell culture is an effective technology for taxol production. This paper discusses the effect of Taxus cell cultures on the 18S rRNA gene sequences based on the phylogenetic analysis of cultured T. chinensis cells and related species. The phylogenetic tree is reconstructed using the maximum parsimony method and the relative rate test to test the hypothesis of a molecular clock. The phylogenetic analysis indicates that cell culture changes the phylogenetic position of cultured T. chinensis cells. More than that, the 18S rRNA gene of cultured T chinensis cells has a faster rate of substitution than that of T. chinensis. With T. media as reference, the divergence time of the cultured T. chinensis cells is 7 Ma (million years) more than that of the T. chinensis cells based on the 18S rRNA gene sequences.     

Nucleolar protein Spindlin1 recognizes H3K4 methylation and stimulates the expression of rRNA genes

The tandem Tudor-like domain-containing protein Spindlin1 has been reported to be a meiotic spindle-associated protein. Here we report that Spindlin1 is not associated with the spindle in mouse embryonic fibroblast cells during mitotic divisions. In interphase cells, Spindlin1 specifically localizes to the nucleoli. Moreover, Spindlin1 is a histone methylation effector protein that specifically recognizes H3K4 methylation. Finally, Spindlin1 localizes to the active ribosomal DNA (rDNA) repeats, and Spindlin1 facilitates the expression of rRNA genes.