Hamish M. Keir

Uptake and excretion of polyamines from baby hamster kidney cells (BHK-21/C13) the effect of serum on confluent cell cultures

In confluent cultures of BHK-21/C13 cells there was little uptake of exogenous polyamines and only a low level of polyamine biosynthesis. These cultures continuously excreted polyamines into the extracellular medium. Spermidine, in both the free and bound form, was the predominant excretion product, whereas the major intracellular polyamine was spermine implying that excretion of polyamines was specific. Reinitiation of growth by the addition of fresh serum immediately increased the uptake of exogenous putrescine, increased the biosynthesis of polyamines and decreased the excretion of polyamines. Thus, polyamine transport into and out of the cell appears to be regulated by the growth status of that cell.

Polyamine metabolism in BHK21/C13 cells: Loss of spermidine from cells following transfer to serum-depleted medium

Abstract The growth rate of BHK21/C13 cells in culture was slowed down by transferring growing cells to serum-depleted medium Following deprivation of serum, the intracellular concentration of polyamines decreased. The amount of spermidine relative to spermine decreased, and this change was the result of the spermidine content per cell decreasing more than the spermine content. The decrease in cell content of polyamines was accompanied by release of polyamines from the cells into the culture medium. The polyamines released were examined using cells whose polyamines had been labelled by prior incubation of the cells with radioactive putrescine. Almost all of the radioactivity released into the medium was found in spermidine, even though the cells contained most of their radioactivity in spermine. It is suggested that specific release of spermidine may be an important mechanism by which these cells can regulate their intracellular content of polyamines.

Factors affecting polyamine excretion from mammalian cells in culture. Inhibitors of polyamine biosynthesis.

Canavanine, diaminopropane, α‐methylornithine and methylglyoxal bis(guanylhydrazone) decreased the intracellular polyamine concentrations in growing baby hamster kidney cells. Each of the inhibitors also prevented polyamine efflux into the extracellular medium. Concomitant with the decrease in polyamine excretion was a change in the distribution of polyamines in the extracellular medium. In each case there was a decrease in the amount of radioactivity present as free spermidine and an increase in that found as acetyl polyamines. The magnitude of this shift correlated with the degree of inhibition of excretion. It may be that acetyl polyamines play a role in the regulation of polyamine excretion.

Subcellular location and growth stage dependence of the DNA polymerases of Euglena gracilis.

The subcellular location and growth stage dependence of the DNA polymerases of Euglena gracilis strain Z and of a bleached derivative of the strain have been studied by fractionation of the enzymes from extracts of whole cells and subcellular fractions on DEAE-cellulose. A new method for the rapid isolation of nuclei was employed. Of the major enzymes, pol A has a predominantly nuclear location and pol B a predominantly cytoplasmic location. Pol A is 4-fold and pol B 15-fold more active in exponentially-growing cells than in stationary-phase cells, pol B representing 90% of the combined activities in exponential-phase cells. The activity of the mitochondrial DNA polymerase increases about 3-fold as the cells enter stationary phase while that of the chloroplast DNA polymerase is greater in exponential-phase cells. The chloroplast enzyme persists in cells which have been reversibly bleached. The results are compared to those of similar experiments involving primitive and higher eucaryotes.

The Effect of Polyamines on Herpes Simplex Virus Type 1 DNA Polymerase Purified from Infected Baby Hamster Kidney Cells (BHK-21/C13)

The DNA polymerase whose synthesis is directed by the herpes simplex virus type 1 (HSV-1) DNA was purified 545-fold from BHK-21/C13 cells 16 h after infection with the virus. Spermidine and spermine stimulated the activity of the polymerase over the concentration range 0.5 mM to 2.5 mM. This effect was enhanced with increased concentrations of polyamine, maximum stimulation being threefold and fourfold for spermidine and spermine respectively. The diamine, putrescine, had little effect on the enzyme at the concentrations used (0.25 to 1.25 mM).

Excretion of Polyamines from Baby Hamster Kidney Cells (BHK-21/C13): Effect of Infection with Herpes Simplex Virus Type 1

Confluent monolayers of BHK-21/C13 cells excreted increasing amounts of polyamines into the extracellular medium with time. Excretion was specificity of spermidine and was not the result of cell death or lysis. Herpes simplex virus type 1 (HSV-1) completely prevented excretion of polyamines from 2 h after infection of the cells. The virus specificity inhibited the release of free spermidine but not of conjugated polyamines.

Effect of spermine on the activity of herpes simplex virus type 1 DNA polymerase: Influence of the template

1. Introduction The polyamines, putrescine, spermidine and sper- mine are cellular polycations whose intracellular con- centrations appear to be important in the regulation of macromolecular synthesis and growth of many types of cells and viruses [ 1,2]. Putrescine and the related polyamines have been shown to have marked growth-stimulating activity in several cell systems including Chinese hamster cells [3] and human embryonic fibroblasts [4]. Similarly, some DNA viruses such as vaccinia, human cytomegalovirus and herpes simplex virus (HSV) have an obligate require- ment for polyamines for virus production [ 5-71. The site of action of the polyamines in cells and viruses is not known, although they may be necessary for DNA replication [8,9]. For example, treatment of lymphocytes with methylglyoxal bis(guanylhydra- zone) or a-methylornithine, both of which inhibit the biosynthesis of polyamines, resulted in specific inhi- bition of DNA synthesis. Since neither drug had any effect on DNA synthesis in vitro, and since the degree of inhibition of DNA synthesis correlated with the degree of polyamine deficiency, it was concluded that DNA replication was one cellular site of action of the polyamines [9]. Similarly in work with viruses evi- dence from studies in vitro has shown that poly- amines optimise the synthesis of HSV-DNA [lo] and + Present

The effect of polyamines on DNA synthesis in vitro

Nuclei and DNA-dependent polymerases alpha and beta were isolated from exponentially growing baby hamster kidney 21/C13 cells and were used to study the effects of polyamines on DNA synthesis in vitro. The greatest effect was observed with spermine, which inhibited both nuclear DNA synthesis and the activity of partially purified DNA polymerase alpha. At 2.5 mM spermine, the maximum concentration used, we observed 58 and 68% inhibition of DNA synthesis by isolated nuclei and polymerase alpha, respectively. In contrast, spermidine caused a small increase in nuclear DNA synthesis at low concentrations (0.5 mM) and inhibition at higher concentrations (2.5 mM); it had no significant effect on the partially purified polymerase alpha. Neither polyamine had any appreciable effect on polymerase beta activity. The results are consistent with the concept that DNA polymerase alpha catalyses the observed DNA synthesis in isolated nuclei.

Multiple ribonuclease H activities from BHK-21/C13 cells

Ribonuclease H, first discovered in calf thymus [ 1 ] , specifically degrades the RNA component of DNARNA hybrid molecules. It has since been isolated from other sources including KB cells [2] , Escherichia coli [3,4] and purified preparations of RNA-dependent DNA polymerase from avian myeloblastosis virus [4,5]. Two ribonuclease H activities have been isolated from rat liver [6] and from the murine myeloma MOf’C-21 [7]. Current evidence [8-l 21 suggests that DNA synthesis may be initated by RNA, in both prokaryotic and eukaryotic systems. Our interest in ribonuclease H stems from (a) the putative role that the enzyme has in DNA replication, selectively removing initiating RNA strands, and(b) our observation that purified preparations of DNA-dependent DNA polymerases I and If from BHK-2l/Cl3 cells contain ribonuclease H activity [ 131. This paper describes multiple forms of ribonuclease H in BHK-2 1 /C 13 cells.

Some observations on the cofactor requirement for partially purified DNA ligase from Escherichia coli

Polynucleotide ligase (DNA ligase) catalyses the synthesis of a phosphodiester bond by esterification of the 5’-phosphoryl group to tlie3’;hydroxyl group of DNA chains which have been properly aligned in a double helical structure. Although the DNA substrate specificity is the same for all ligases studied, the cofactor requirement for the reaction does vary depending on the source of the enzyme. Escherichia coli ligase specifically requires NAF whereas the specificity of T4 bacteriophage-induced and mammalian ligases is for ATP [l]. In the course of investigating mammalian cell ligase we decided to check a simple ligase assay system using enzyme prepared from E. coli. This paper reports our findings that partially purified ligase from E. coZi C641 catalysed the covalent closure of “Hershey” circles [2] of h DNA only in the presence of ATP and that NAF was not required for the reaction. The requirement for ATP was lost during purification of the ligase, and after the final stage of purification NAITcould be shown to increase the amount of covalently closed product. The possibilities of exceptionally high levels of ligase-AMP in the bacterial cell, a protective effect of ATP on the DNA substrate and a “modification” of the ligase during purification are discussed.