Changes in the abundance of polyadenylated RNA during slime mould development measured using cloned molecular hybridization probes.

Total Dictyostelium discoideum messenger RNA prepared from cells at the eighth hour of development in suspension culture has been copied into DNA. This DNA was inserted into the plasmid PMB9 and used to transform Escherichia coli. The resulting “clone bank” was screened using an in situ hybridization technique in which replicate copies of a set of clones were hybridized with mRNA isolated from vegetative (non-developing) cells and from cells at the eighth hour of development. The mRNA was labelled in vitro so that the amount of hybridization to a given clone is a measure of the relative abundance of the mRNA complementary to the DNA in that clone. By comparing the amount of hybridization of the mRNA preparations to each clone, it has been possible to identify plasmids containing D. discoideum DNA whose complementary mRNA increases or decreases in abundance during development. These observations are direct proof of a change in mRNA concentration during D. discoideum development for individual high and medium abundance mRNA species. We can estimate from these results the proportion of such mRNA species whose concentration increases significantly during development and we find that only a small fraction show such a change.     

Contact-dependent regulation of vinculin expression in cultured fibroblasts: a study with vinculin-specific cDNA probes.

Vinculin specific cDNA clones were isolated from chicken embryo fibroblast (CEF) cDNA library in lambda gt11. The clones, ranging in size from 2.8 to 5.0 kb, were initially selected by rabbit antibodies to vinculin. Their identity was further confirmed by their specific reactivities with a battery of different vinculin-specific monoclonal antibodies. Southern blot analysis of restriction enzyme digested chicken spleen DNA suggested that all the isolated cDNA clones correspond to the same gene(s). Northern blot hybridization revealed that the vinculin-specific cDNA clones react with a single 6.5 kb mRNA in total cellular RNA preparations of CEF, whole chicken embryos and chicken gizzard smooth muscle. Moreover, fractionation of CEF poly(A)+ RNA by sucrose gradient centrifugation followed by translation in cell free system indicated that the mRNA coding for vinculin has a size of about 6.0-7.0 kb. The identity of these clones was finally confirmed by selection hybridization assay. The isolated vinculin-specific cDNA probes were subsequently used in order to study the effect of substrate adhesiveness on the expression of vinculin. We show here that cells cultured on highly adhesive substrate, such as endothelial extracellular matrix (ECM), form large vinculin-rich focal contacts, while cells grown on poorly adhesive substrate poly(2-hydroxyethyl methacrylate) [poly(HEMA)] contain only small distorted vinculin spots. These morphological differences were accompanied by over 5-fold reduction in vinculin synthesis in cells growing on poly(HEMA), compared to those cultured on the ECM and over 7.5-fold decrease in the levels of vinculin-specific mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Re-modelling of nuclear architecture in quiescent and senescent human fibroblasts

Spatial organisation of the genome within the nucleus can play a role in maintaining the expressed or silent state of some genes [1]. There are distinct addresses for specific chromosomes, which have different functional characteristics, within the nuclei of dividing populations of human cells [2]. Here, we demonstrate that this level of nuclear architecture is altered in cells that have become either quiescent or senescent. Upon cell cycle exit, a gene-poor human chromosome moves from a location at the nuclear periphery to a more internal site in the nucleus, and changes its associations with nuclear substructures. The chromosome moves back toward the edge of the nucleus at a distinctive time after re-entry into the cell cycle. There is a 2-4 hour period at the beginning of G1 when the spatial organisation of these human chromosomes is established. Lastly, these experiments provide evidence that temporal control of DNA replication can be independent of spatial chromosome organisation. We conclude that the sub-nuclear organisation of chromosomes in quiescent or senescent mammalian somatic cells is fundamentally different from that in proliferating cells and that the spatial organisation of the genome is plastic.     

Enhanced transcription in a pre-B-like cell line transformed with cloned antibody genes after antigen-specific stimulation

The transducing vector, pSV2-neo, carrying the rearranged immunoglobulin (Ig) heavy (mu) and light (kappa) chain genes specific for the hapten 2,4,6-trinitrophenyl (TNP) was introduced into a pre-B cell line. The transformants expressed the TNP-specific IgM receptor on the surface. Furthermore, the addition of TNP-bovine serum albumin (hapten-carrier conjugate) to the culture media activated the expression of the transferred Ig genes and several endogenous genes such as v-abl and beta-tubulin. However, expression of the beta2-microglobulin gene was not affected. The results presented in this paper show that transfection of cloned Ig genes into B cells is a useful system for establishing monoclonal B cell lines expressing functional Ig receptor molecules with defined hapten specificity.     

Functional expression of the polymeric immunoglobulin receptor from cloned cDNA in fibroblasts

The polymeric immunoglobulin receptor, a transmembrane protein, is made by a variety of polarized epithelial cells. After synthesis, the receptor is sent to the basolateral surface where it binds polymeric IgA and IgM. The receptor-ligand complex is endocytosed, transported across the cell in vesicles, and re-exocytosed at the apical surface. At some point the receptor is proteolytically cleaved so that its extracellular ligand binding portion (known as secretory component) is severed from the membrane and released together with the polymeric immunoglobulin at the apical surface. We have used a cDNA clone coding for the rabbit receptor and a retroviral expression system to express the receptor in a nonpolarized mouse fibroblast cell line, psi 2, that normally does not synthesize the receptor. The receptor is glycosylated and sent to the cell surface. The cell cleaves the receptor to a group of polypeptides that are released into the medium and co-migrate with authentic rabbit secretory component. Cleavage and release of secretory component do not depend on the presence of ligand. The cells express on their surface 9,600 binding sites for the ligand, dimeric IgA. The ligand can be rapidly endocytosed and then re-exocytosed, all within approximately 10 min. Very little ligand is degraded. At least some of the ligand that is released from the cells is bound to secretory component. The results presented indicate that we have established a powerful new system for analyzing the complex steps in the transport of poly-Ig and the general problem of membrane protein sorting.     

Increased ouabain-sensitive 86Rubidium uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplication-stimulating activity

Multiplication-stimulating activity (MSA), a protein which stimulates DNA synthesis and growth of chicken embryo fibroblasts, was purified from serum-free medium conditioned by the growth of a rat liver cell line. Purified MSA was shown to rapidly stimulate ouabain-sensitive Na+, K+-ATPase activity as measured by both enzyme assay and rate of 86Rubidium uptake. Labeled ouabain binding was also shown to increase after stimulation of quiescent cells by serum or purified MSA. Conditions which interfere with the ability of the cells to accumulate potassium, such as the presence of the specific inhibitor, ouabain; incubation in potassium-free medium; or the presence of the potassium ionophore, valinomycin, were all demonstrated to inhibit the stimulation of DNA synthesis by serum or purified MSA. These results suggest that an early event in the stimulation of DNA synthesis by purified MSA is an activation of membrane Na+, K+-ATPase with a resulting accumulation of potassium ions inside the cell.     

Heterogeneity of the changes in cytoplasmic pH upon serum stimulation of quiescent fibroblasts

Addition of mitogens to quiescent cells results in rapid ionic changes in the cytoplasm, including pH. We studied the changes in cytoplasmic pH in single Swiss 3T3 cells upon serum stimulation using fluorescence ratio imaging microscopy. Quiescence was attained using two approaches, serum deprivation of subconfluent cells and confluence. All measurements were made in the presence of bicarbonate and the absence of other organic buffers. We also used BCECF coupled to dextran to avoid several artifacts associated with using BCECF-AM, including leakage and phototoxicity. Analysis of the changes in cytoplasmic pH demonstrated a dramatic heterogeneity in the responses of single cells. There were six basic classes of responses, 1) a fast alkalinization, reaching a maximum pH in approximately 2-5 min; 2) a slow alkalinization, reaching a maximum pH in 10-20 min; 3) a very slow alkalinization, not reaching a plateau pH within the measurement time; 4) no apparent change in pH during the measurement time; 5) an early transient acidification, followed by either a fast or slow alkalinization; and 6) an acidification, followed by alkalinization and then by a decrease to some intermediate pH. Subconfluent cells exhibited greater heterogeneity in response than confluent cells, with no single dominant class of response. The dominant (55%) response for confluent cells was a gradual alkalinization of approximately 0.01 pH units/min. A larger proportion (52%) of subconfluent cells exhibited an early transient acidification compared to confluent cells (7%). A significant proportion of both types of cells (23% subconfluent, 36% confluent) exhibited no change in cytoplasmic pH upon stimulation. In general, the kinetics of changes in cytoplasmic pH were significantly different from the published results with population averaging methods.     

Changes in RNA metabolism and accumulation of presumptive messenger RNA during transition from the growing to the quiescent state of cultured mouse fibroblasts.

Mouse fibroblasts maintained in tissue culture regulate total protein and ribosomal RNA synthesis co-ordinately with changes in the cellular growth state. Here we show that changes in the rate of synthesis of nuclear non-polyadenylic acid-containing RNA and the rate of accumulation and breakdown of cytoplasmic ribosomal RNA also accompany the transition from the resting to the growing cellular growth state, while the rate of synthesis of nuclear poly (A)-containing RNA and the rates of accumulation and breakdown of cytoplasmic poly(A) containing RNA (presumptive messenger RNA) are, however, only marginally changed. The small net increase (20% to 30%) in the amount of presumptive mRNA is considerably less than the observed increase in protein synthesis (two to threefold) during this transition. We also isolated and characterized extra-polysomal poly(A)-containing ribonucleoprotein particles from quiescent cultures that were similar to those particles obtained by treatment of polyribosomes with EDTA. These experiments suggest that the early increase in protein synthetic activity when quiescent, cultured cells are induced to grow is partially caused by an increased attachment of pre-existing mRNA molecules to free ribosomes.     

The isolation of cloned cDNA sequences which are differentially expressed in human lymphocytes and fibroblasts.

Poly(A)+ RNA populations derived from normal lymphocytes and fibroblasts have been compared by hybridising each RNA to cDNA derived from the other RNA population. This indicated that approximately 75% of the sequences were common to both, and that these were present at different concentrations in the two cell types. The two RNA populations were further compared by hybridising them to a cDNA recombinant library derived from lymphocyte poly(A)+ RNA. This allowed the identification of clones containing sequences which are abundant in lymphocyte poly(A)+ RNA but absent or rare in fibroblast poly(A)+ RNA. A direct estimation of the abundance of five of these sequences in lymphocyte cDNA demonstrated that clones can be detected by such a procedure if they represent 0.2% or greater of the original cDNA population.     

Enhanced synthesis and extracellular accumulation of hyaluronic acid during stimulation of quiescent human fibroblasts by mouse epidermal growth factor

The effect of mouse epidermal growth factor (mEGF) on the synthesis of glycosaminoglycans and glycoproteins by human fibroblasts has been studied. The addition of physiological concentrations (10^?9 M) of mEGF to quiescent cultures preincubated in the absence of serum was found to elicit an increased incorporation of ³H-glucosamine into the glycosaminoglycans and glycoproteins of both the cellular and extracellular fractions. Although the growth response to the factor, as measured by DNA replication, was minimal under these conditions as compared with the effect of serum, the mEGF-induced incorporation of glucosamine into these cellular constituents and into the extracellular glycoproteins was comparable to that elicited by serum shift-up. Serum, however, caused a significantly larger incorporation of glucoasmine into extracellular, acid-soluble glycosaminoglycans, which were shown to contain hyaluronic acid as the major component. As previously demonstrated, the growth response to mEGF can be enhanced several fold by an mEGF-binding arginine esterase, which is normally associated with the factor in vivo, and by ascorbate. The esterase was found to increase markedly the mEGF-induced incorporation of glucosamine into extracellular hyaluronic acid, while the addition of ascorbic acid did not significantly alter glucosamine incorporation.