Introns are inconsequential to efficient formation of cellular thymidine kinase mRNA in mouse L cells.

TK mRNA levels were determined in mouse L cells transformed with intron deletion mutations of the chicken TK gene. Whether normalized per cell, per integrated gene, or per internal control signal, intron deletion did not diminish the efficiency of TK mRNA formation in transformed L cells. The results demonstrated that introns are not required for efficient biogenesis of cellular mRNA in transformed mouse L cells.     

Induction of thymidine kinase and DNase in varicella-zoster virus-infected cells and kinetic properties of the virus-induced thymidine kinase.

Thymidine kinase (TK), DNA polymerase, and DNase activities were induced in human foreskin fibroblasts after varicella-zoster virus infection. The induced TK and DNase activities have electrophoretic mobilities different from the corresponding host enzymes. Varicella-zoster virus-induced TK was purified and separated from the host enzyme by affinity column chromatography. This enzyme has been shown to have a broader substrate specificity with respect to either the phosphate donor or acceptor as compared with human cytoplasmic and mitochondrial TKs. The best phosphate donor is ATP, with a Km of 16 microM. The Km values of thymidine, deoxycytidine, and 5-propyl deoxyuridine were estimated to be 0.4, 180, and 0.8 microM, respectively. The Ki values for several analogs of thymidine such as 5-iododeoxyuridine, arabinofuranosylthymine, 5-ethyl deoxyuridine, and 5-cyanodeoxyuridine were also examined. TTP acted as a noncompetitive inhibitor with respect to thymidine with a Ki of 5 microM. The kinetic behavior of varicella-zoster virus-induced TK is different from human cytoplasmic, human mitochondrial, and herpes simplex virus type 1- and 2-induced TKs.     

Cardiac mechanics at the cellular level.

The active mechanical properties of heart muscle are load, length, and time-dependent. The capability for investigating cardiac mechanisms at the cellular level may help to distinguish between those properties of the myocardium which arise from myocardial cells and those which arise from the tissue architecture and extracellular matrix of connective fibers. We present here, for the first time, a general approach for subjecting single heart cells to isometric, isotonic, afterloaded, or physiological loading sequences, while obtaining on-line measures of cell force and length. This approach has been implemented and tested on freshly dissociated, adult frog ventricular myocytes. Examples are presented for each of the four loading sequences.     

Induction of thymidine kinase in enzyme-deficient chinese hamster cells

Previous work with Chinese hamster cells suggests that thymidine kinase deficiency and loss of potential for plating in HAT medium may arise by a process of mutation coupled with site-specific repression by bromodeoxyuridine at the tk locus. In this study, tk^? Chinese hamster cells were exposed to a series of inductors to determine whether revertants for the putative second stage originate by genetic or epigenetic change. Brief exposure to 5-azacytidine resulted in massive conversion to the HAT⁺ state, and revertants showed levels of thymidine kinase activity intermediate between those of tk^? and wild-type cells. By contrast, incidence of HAT⁺ cells rose only slightly in populations mutagenized with ethyl methanesulfonate. Large increases in frequency of HAT⁺ cells were obtained by treatment with n-butyrate and L-ethionine, which affect gene expression in other cell systems but have no known mutagenic potential. Induction of HAT⁺ revertants seems to be mediated by a stable epigenetic shift, which reverses the gradual extinction of thymidine kinase activity in the parent cells. The data support the view that induction in Chinese hamster cells results from changes in DNA methylation patterns, and suggest studies to define the process in molecular terms.     

Independent regulation of thymidine kinase mRNA and enzyme levels in serum-stimulated cells.

We have studied the regulation of thymidine kinase mRNA and protein/enzyme expression in quiescent and serum-stimulated rat cells transfected with a human TK cDNA clone expressed from a number of promoters. Our results indicate that while the pattern of mRNA expression is a function of the promoter used, the pattern of protein/enzyme expression is not. When the gene is expressed from the homologous human TK promoter both mRNA and enzyme levels remain low throughout G1 and increase as the cells enter S phase. When it is expressed from the heterologous SV40 early promoter, mRNA levels are high throughout G1, but enzyme and protein levels remain low until 8-10 h following serum stimulation. Thus, protein levels appear to be uncoupled from mRNA levels in this system, suggesting the presence of translational and/or posttranslational regulation. An analysis of mutant cDNA clones indicates that this regulation is not dependent upon sequences at the 5' or 3' end of the cDNA, including the entire 5'-untranslated region, the authentic AUG and the first 48 nucleotides of the coding region.     

Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences.

We have constructed a chimeric thymidine kinase (TK) minigene, pHe delta 6Ha, which combines the complete coding and 3' noncoding regions of a Chinese hamster TK cDNA with the promoter region and 5' untranslated region of the TK gene of herpes simplex virus type 1. We have transformed rat 4 cells to Tk+ with this gene and analyzed the pattern of TK gene expression in these transformants under various conditions of in vitro cell culture. We find that TK gene expression in these Tk+ transformants is growth phase dependent, responsive to adenovirus 5 infection, and indistinguishable in character under a variety of cell culture conditions from the pattern of TK gene expression in rat 4 cells transformed to Tk+ with the genomic Chinese hamster TK gene clone lambda HaTK.5. We are led to the conclusion that the genetic elements which mediate growth phase-dependent TK gene expression are contained entirely within the sequences of the mature cytoplasmic hamster TK mRNA.     

Induction of fetal thymidine kinase by phyto-estrogens in the immature rat uterus

Administration of phytooestrogens to immature female rats leads to a large increase in uterine thymidine kinase activity. That increase concerns to a large extent the fetal isoenzyme of thymidine kinase. These results confirm the estrogenic properties of phytoestrogens and allow to specify their physiological effects.     

Rhodopsin phosphorylation occurs at metarhodopsin II level

Photolyzed rhodopsin was phosphorylated in bovine rod outer segments incubated at –10 C. In the experiment in which urea-treated outer segments and rhodopsin kinase were incubated with ATP in the presence of 30% glycerol, the extent of phosphate incorporation at –10 C was about 30% of that at 37 C. Separation of phosphorylated rhodopsin by isoelectric focusing indicated that a limited number of sites were phosphorylated at –10 C. The partially phosphorylated pigment incorporated more phosphates when the temperatures was raised to 37 C. This was partly due to decreased inhibition of phosphorylation by glycerol at higher temperature. Since the maximum phosphorylation at –10 C (at which metarhodopsin II is stable) occurred at a pH value (6.0) lower than the pKa for metarhodopsin I-metarhodopsin II transition, metarhodopsin II was suggested to be the preferred substrate for rhodopsin kinase at –10 C. Limited proteolysis with thermolysin of rhodopsin phosphorylated at 37 C released peptides containing about 50% of the total phosphate incorporated. In contrast, proteolytic digestion of rhodopsin phosphorylated at –10 C released negligible amounts of phosphate-containing peptides. The results were taken to suggest that the incorporation of phosphates at metarhodopsin II level under the present condition occurred in the residues other than those removed by thermolysin digestion.Based on material presented at the Fifth International Congress of Eye Research, Eindhoven, October 1982     

Epstein-Barr virus-associated thymidine kinase.

Superinfection of Raji cells with Epstein-Barr virus induced a new thymidine kinase that was distinguishable from both adult and fetal kinases of the host cell by discontinuous electrophoresis on polyacrylamide gels and glycerol gradients.     

Baroreceptor mechanisms at the cellular level

Nothing is known of transduction mechanisms of baroreceptors in vivo. Not even the site of transduction is known. However, there are mechanotransducer ion channels that provide a useful model system of transduction. In these channels, transduction is accomplished by a strain-dependent increase in the probability of being open. Membrane tension is coupled to the channel by cytoskeletal strands that concentrate the strain energy from a large (approximately equal to 4000 A diameter) area of membrane and thereby provide high sensitivity. The channel is fast and does not inactivate, but viscoelastic coupling to the channel can dramatically alter the transfer function.