Search

Polyamines, in particular spermidine and spermine, were shown to have a stimulatory effect on the incorporation of amino acids into protein in the wheat-germ cell-free system programmed with exogenous mRNA. The addition of the optimum concentrations of polyamines results in a lowering in the Mg2+ optimum for protein synthesis. At the optimum combination of spermidine and Mg2+ the rate of peptide chain elongation with tobacco mosaic virus RNA as messenger RNA was twice the rate observed at the optimum concentration of Mg2+ in the absence of polyamines, but there was no significant difference in the rate of chain initiation under these two conditions. The presence of polyamines also increased the yield of full-length translation products from the viral RNA with a corresponding reduction in the short polypeptide products. These short products are shown to be incomplete translation products that accumulate mainly in the form of peptidyl-tRNA associated with 80-S ribosomes which can release the incomplete nascent chains on incubation with puromycin. The origin of these incomplete chains may be due to endonucleolytic cleavage of mRNA, which we have demonstrated occurs at a significant rate in our system. It is proposed that the increased rate of elongation in the presence of polyamines raises the probability of ribosomes completing the synthesis of full-length products before the mRNA is degraded.

A so-called 'growth-associated' or 'M-phase specific' histone H1 kinase (H1K) has been described in a wide variety of eukaryotic cell types; p34cdc2 has previously been shown to be a catalytic subunit of this protein kinase. In fertilized sea urchin eggs the activity of H1K oscillates during the cell division cycle and there is a striking temporal correlation between H1K activation and the accumulation of a phosphorylated form of cyclin. H1K activity declines in parallel with proteolytic cyclin destruction of the end of the first cell cycle. By virtue of the high affinity of the fission yeast p13suc1 for the p34cdc2 protein, H1K strongly binds to p13-Sepharose beads. Cyclin, p34cdc2 and H1K co-purify on this affinity reagent as well as through several conventional chromatographic procedures. Anticyclin antibodies immunoprecipitate the M-phase specific H1K in crude extracts or in purified fractions. Sea urchin eggs appear to contain much less cyclin than p34cdc2, suggesting that p34cdc2 may interact with other proteins. These results demonstrate that cyclin and p34cdc2 are major components of the M-phase specific H1K.

Cleavage in embryos of the sea urchin Arbacia punctulata consists of eight very rapid divisions that require continual protein synthesis to sustain them. This synthesis is programmed by stored maternal mRNAs, which code for three or four particularly abundant proteins whose synthesis is barely if at all detectable in the unfertilized egg. One of these proteins is destroyed every time the cells divide. Eggs of the sea urchin Lytechinus pictus and oocytes of the surf clam Spisula solidissima also contain proteins that only start to be made after fertilization and are destroyed at certain points in the cell division cycle. We propose to call these proteins the cyclins.

This paper reviews the evidence that protein synthesis in rabbit reticulocytes is regulated by the reversible phosphorylation of the initiation factor eIF-2 by protein kinases under the control of the cytoplasmic haemin concentration on the one hand, and double-stranded RNA on the other. A molecular mechanism is proposed to account for the observation that inhibition of protein synthesis occurs when considerably less than half the eIF-2 present has been phosphorylated. The question of whether phosphorylation regulates protein synthesis in other types of cell is discussed.

1 A coupled transcription and translation system is described in which protein synthesis is directed by mRNA synthesised in situ by vaccinia virus cores. The cell-free system is based on a micrococcal-nuclease-treated reticulocyte lysate. 2 The polypeptides made in vitro include many authentic early vaccinia proteins, but also other proteins which were not detected in infected cells. 3 Concentrations of cores which inhibit host cell protein synthesis in vivo caused a delayed inhibition of translation in vitro; this was partly, but not entirely, due to dsRNA associated with the cores. 4 The mRNA made was methylated by core enzymes. Inhibition of methylation reduced the rate of translation tenfold; unmethylated RNA bound ribosomes poorly, but was nevertheless translated faithfully.

Single-stranded cDNA clones complementary to the 5' end of TMV RNA have been used to explore the conditions necessary for efficient 'hybrid arrest of translation' in the reticulocyte lysate. It is shown that incubations of 20 minutes at 60 degrees in 0.1 M KCl are sufficient to give almost complete arrest of translation using a clone complementary to the 5'-non-coding region and first 171 coding nucleotides of TMV RNA. However, hybrids with DNA complementary to regions of the mRNA downstream of the first AUG gave variable and in some cases almost no arrest of translation in the reticulocyte lysate unless they were first digested with RNase H. A simple and rapid method for giving complete and highly specific arrest of translation of particular mRNAs in complex mixtures has been developed using both cDNA clones and synthetic oligodeoxynucleotides in conjunction with RNase H digestion. Evidence is presented that suggests that 'hybrid arrest of translation' in the wheat-germ cell-free system is primarily due to the action of RNase H. When a reticulocyte lysate was doped with 20 U/ml of RNase H, its ability to translate unannealed mRNA was unaffected but it translated DNA/RNA hybrids extremely poorly.

Publisher Summary This chapter examines the control of protein synthesis in rabbit reticulocyte lysates. Reticulocytes need iron to make globin, and the supply of hemin regulates protein synthesis in these cells. A simple experiment has shown that the inhibition of protein synthesis occurs because of the activation of an inhibitor rather than some initiation factor requiring hemin as an essential cofactor in these particular cells. Subsequent studies showed that the inhibitor was a protein that acted catalytically; one molecule could inhibit over 100 ribosomes. They also defined a pathway of activation of the inhibitor called hemin controlled repressor (HCR) showing that it normally existed as an inactive entity, which could be activated either by incubation at physiological temperature in the absence of hemin or by brief incubation with N-ethylmaleimide or other –SH reactive reagents. At early stages of activation, it can be deactivated by the addition of hemin, but at later stages, hemin has no effect on it.

Using a translation assay we have shown that the axoplasm of the squid giant axon contains significant amounts of mRNA coding for a heterogeneous group of prot sets of proteins specified by glial and neuronal perikaryal mRNA. Messenger RNA is associated with the "microsomal" fraction of the axoplasm. The possible involvement of axoplasmic mRNA in protein synthesis remains to be ascertained. It is known that axoplasmic proteins are synthesized by the isolated giant axon, presumably by the surrounding glia cells.

Summary In cleaving Xenopus eggs, the cell division cycle is abbreviated to a rapid succession of S and M phases. During mitosis a number of proteins show increased phosphorylation due to the activation of a histone H1 kinase, the homologue of the cdc2+ gene product of the yeast Schizosaccharomyces pombe. We have studied the regulation of the activity of this enzyme in cell-free extracts of Xenopus eggs. In extracts of activated eggs incubated at 22°C, histone H1 kinase activity shows two peaks of activation and disappearance. Activation occurs in two stages. The first stage requires protein synthesis, whereas the second does not. The second stage of activation involves post-translational activation of the kinase. Kinase activity rises to a peak and then abruptly disappears. Added sea urchin cyclin is degraded at the time of disappearance of kinase activity. The oscillation in kinase activity is then repeated, usually with lower amplitude. Post-translational activation of the kinase requires a membrane-containing particulate cellular component, whose role has yet to be defined. The kinase can still be activated in the presence of EDTA or in the presence of the ATP analogue, 6-dimethylaminopurine, which implies that phosphorylation of the kinase complex is not required for activation. Under these conditions, however, the kinase activity does not show its normal sudden disappearance, and added cyclin is perfectly stable. These observations are consistent with the idea that post-translational activation of the kinase involves protein phosphatase activity, whereas switching off the kinase requires an ATP–Mg2+ -dependent reaction, perhaps due to protein phosphorylation.

When rabbit reticulocyte lysates were gel-filtered on Sephadex G-25 or G-50, the rate of protein synthesis in the gel-filtered lysate was the same as in the parent lysate provided appropriate concentrations of ATP, GTP and spermidine or spermine were added. The polyamines were found to increase the rate of synthesis and to lower the Mg2+ optimum. Although gel-filtered lystes prepared in this way syntheses protein at a high initial rate, this rate is maintained for only about 20 min, after which it declines rapidly to a very low rate, even though optimal concentrations of haemin are present. This shut-off was completely prevented by the addition of low concentrations of sugar phosphates capable of acting as NADPH generators: glucose 6-phosphate, 2-deoxyglucose 6-phosphate, ribulose 5-phosphate and ribose 5-phosphate. Although the addition of isocitrate resulted in the generation of NADPH, the stimulation of protein synthesis was normally less than was achieved with glucose 6-phosphate. Dithiothreitol also promoted only partial activation, as did fructose 1,6-bisphosphate, a sugar phosphate which does not generate NADPH in this system. Full stimulation was observed, however, when both fructose 1,6-bisphosphate and either dithiothreitol or isocitrate were added. It is argued that the maintenance of maximum rates of protein synthesis in gel-filtered lysates requires the presence of both a sugar phosphate and a reducing agent, which can be dithiothreitol or an NADPH-generating system. Low concentrations of thioredoxin were shown to be required for the stimulatory effect of NADPH-generating systems, but not for stimulation by dithiothreitol. On the basis of these studies, a recommended procedure is given for the preparation of highly active gel-filtered lysates and gel-filtered nuclease-treated lysates.

In both clam oocytes and sea urchin eggs, fertilization triggers the synthesis of a set of proteins specified by stored maternal mRNAs. One of the most abundant of these (p41) has a molecular weight of 41,000. This paper describes the identification of p41 as the small subunit of ribonucleotide reductase, the enzyme that provides the precursors necessary for DNA synthesis. This identification is based mainly on the amino acid sequence deduced from cDNA clones corresponding to p41, which shows homology with a gene in Herpes Simplex virus that is thought to encode the small subunit of viral ribonucleotide reductase. Comparison with the B2 (small) subunit of Escherichia coli ribonucleotide reductase also shows striking homology in certain conserved regions of the molecule. However, our attention was originally drawn to protein p41 because it was specifically retained by an affinity column bearing the monoclonal antibody YL 1/2, which reacts with alpha-tubulin (Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582). The finding that this antibody inhibits the activity of sea urchin embryo ribonucleotide reductase confirmed the identity of p41 as the small subunit. The unexpected binding of the small subunit of ribonucleotide reductase can be accounted for by its carboxy-terminal sequence, which matches the specificity requirements of YL 1/2 as determined by Wehland et al. (Wehland, J., H. C. Schroeder, and K. Weber, 1984, EMBO [Eur. Mol. Biol. Organ.] J., 3:1295-1300). Unlike the small subunit, there is no sign of synthesis of a corresponding large subunit of ribonucleotide reductase after fertilization. Since most enzymes of this type require two subunits for activity, we suspect that the unfertilized oocytes contain a stockpile of large subunits ready for combination with newly made small subunits. Thus, synthesis of the small subunit of ribonucleotide reductase represents a very clear example of the developmental regulation of enzyme activity by control of gene expression at the level of translation.

Publisher Summary The nuclease-treated rabbit reticulocyte lysate supplemented with heterologous tRNA is the most widely used translation system for eukaryotic mRNAs. This chapter describes the procedures to make the lysate, how to make it dependent on added mRNA, and provide some suggestions for methods of preparing gel-filtered nuclease-treated lysates. The ideal translation system is a reticulocyte lysate from which the endogenous mRNA is removed by fractionation or selective destruction without impairing the intrinsic high activity of the translation machinery. The resulting nuclease-treated lysate has a very low activity, unless eukaryotic mRNAs are added. The reticulocytes are highly specialized cells, and the translation machinery shows little specialization or preference with regard to initiation of protein synthesis on different eukaryotic mRNAs. It is only with respect to the complement of tRNA species that the reticulocyte lysate shows special properties, which may impair its ability to translate heterologous mRNA.

A cell-free extract prepared from activated Xenopus eggs by high-speed centrifugation displays one spontaneous cycle of activation and inactivation of histone H1 kinase and MPF activity that is largely attributable to Xenopus p32cdc2. The timing of the oscillation closely follows that observed in intact eggs, is associated with large changes in endogenous protein phosphorylation and depends entirely on post-translational events. The extract can be fractionated into soluble and particulate material, both of which components are required for the oscillatory behaviour. Kinase activation does not require Mg+ ATP, but its rapid inactivation, which coincides with the destruction of cyclin, is inhibited both by EDTA and the protein kinase inhibitor 6-dimethylaminopurine. This suggests that protein phosphorylation is required for cyclin destruction and kinase inactivation.

The pattern of protein synthesis in oocytes of the starfish Marthasterias glacialis changes during 1-methyladenine-induced meiotic maturation. One of the newly synthesized proteins, a major 54-kDa polypeptide, was synthesized continuously after activation but was destroyed abruptly just before appearance of the polar bodies at each meiotic division. This protein thus resembles the cyclin proteins identified in cleaving sea urchin and clam embryos. RNA extracted from oocytes before and after maturation encoded virtually identical polypeptides when translated in the reticulocyte lysate. However there was poor correspondence between the in vitro translation products and the labeling pattern of intact cells. There was no exact in vitro counterpart to the in vivo-labeled cyclin. Instead, a major polypeptide of 52 kDA was seen which appears to be a precursor of the 54-kDa form of cyclin. The 52-kDa polypeptide was identified as cyclin by hybrid arrest of translation. Cyclin mRNA is not translated to a significant extent before oocyte activation and is present in oocytes as a nonadenylated form. It becomes polyadenylated when the oocytes mature. This behavior is also seen in the case of the mRNA for the small subunit of ribonucleotide reductase, another abundant maternal mRNA whose translation is activated at maturation.

After fertilization of sea urchin (Arbacia punctulata) eggs, there is a single prominent alteration in the pattern of protein phosphorylation. In eggs preloaded with 32PO4, a 31,000 Mr protein (rp31) becomes labeled within 4 min of sperm addition. A new steady-state level of rp31 labeling is achieved by 11 min. The rate of protein synthesis in sea urchin zygotes also increases at 8–10 min after fertilization. Protein rp31 corresponds to mammalian ribosomal S6 because it cosediments with 40 S subunits on high salt-sucrose gradients, it is similar to the mammalian protein in Mr and charge, and it becomes phosphorylated during an increase in protein synthesis. The specific activity of phosphorylated rp31 (relative to rRNA) is similar between free 80 S monosomes and polysomes, indicating that rp31 phosphorylation is not sufficient for ribosomal activity. A phosphatase, highly specific for rp31, is present in extracts of eggs and very early embryos. This phosphatase becomes inactive at about the same time that the degree of labeling of rp31 increases in embryos. Evidently a control system that maintains a low level of rp31 phosphorylation is active in sea urchin eggs. Inactivation of this system shortly after fertilization leads to the accumulation of phosphorylated ribosomes.

A new strategy for the purification of rabbit reticulocyte haem-controlled eIF-2α kinase is described, based on the fact that this kinase can be self-phosphorylated in several sites. Incubation of partially purified kinase with ATP changes its behaviour on anion exchangers sufficiently to separate it from almost all contaminating proteins.

Cyclic AMP and a variety of purines are able to overcome the inhibition of the initiation of protein synthesis caused by incubation of the lysate in the absence of added hemin or with double-stranded RNA or oxidised glutathione. These three inhibitions show similar kinetics and are each accompanied by the disappearance of a complex between the 40S ribosomal subunits and met-tRNAf. A translational repressor has been implicated in the inhibition seen in the absence of hemin and we suggest that the link between these three inhibitions is the accumulation of this repressor.

The initial rate of globin synthesis in reticulocyte lysates is close to the steady-state rate in intact reticulocytes, but the amount of globin made by these preparations is low because the initiation of new chains of globin becomes the rate-limiting step shortly after the start of cell-free incubation. The amount of globin made by a given lysate is constant and independent of the initial rate of protein synthesis if that rate is altered by making the incubations at low concentrations of cycloheximide or at different temperatures. There are two exceptions to these general observations: (1) below about 25 °C, the capacity to initiate is much more stable and protein synthesis continues at a constant rate to a level which exceeds the amount made at the initial rate for higher temperatures. (2) In the presence of 3 × 10−5 m-hemin, the initial rate of initiation is preserved for much longer than in the absence of added hemin. Lysates whose rate of protein synthesis has markedly declined may be reactivated by the addition of ribosome-free cytoplasm from a fresh lysate. A few lysates can be reactivated by the addition of hemin, but in this case synthesis resumes only after a lag of a few minutes. From these observations we draw the conclusion that initiation in reticulocyte lysates involves one or more “soluble” initiation factors which are inactivated by protein synthesis above 25 °C and which are stabilized by hemin.

Infection of HeLa cells with poliovirus results in an inhibition of host-cell protein synthesis. Cytoplasmic extracts from infected cells contian an activity that inhibits initiation of protein synthesis in vitro by rabbit reticulocyte lysates. This inhibitory activity has been purified and characterized, and is shown to reside in poliovirus double-stranded RNA. The intact double-stranded molecule is not essential for inhibitory activity.

The rates of translation of the messenger RNA for the α and β chains of rabbit globin have been measured, and it is found that the α chain is translated faster than the β chain. This means that two different classes of messenger RNA are translated at different rates within the same cells.

The nascent peptides attached to the polysomes of rabbit reticulocytes were labelled with [3H]lysine, and the specific activity of the tryptic peptides derived from them measured. The data show that ribosomes are uniformly distributed on mRNA when the cells are incubated under standard in vitro conditions. When cells were incubated in the absence of tryptophan, or when they were exposed briefly to sodium fluoride after the nascent peptides had been labelled, the ribosomes were found at one or other end of the mRNA. From the uniform distribution of the nascent chains in normally incubated cells we conclude that the rate of movement of the ribosomes over all regions of the mRNA is uniform. We are able to deduce the relative number of ribosomes carrying nascent α and β chains. The number of complete chains isolated with the ribosomes varied from 1 7 to 1 60 complete chain/nascent chains. In no case was an excess of complete α chains over β chains found attached to the ribosomes. These results are discussed in relation to the rate limiting step in the assembly of globin.

Haemin enhances the synthesis of all proteins in reticulocyte lysates, including those programmed by added mRNA

Two B-type cyclins, B1 and B2, have been identified in mammals. Proliferating cells express both cyclins, which bind to and activate p34 cdc2 . To test whether the two B-type cyclins have distinct roles, we generated lines of transgenic mice, one lacking cyclin B1 and the other lacking cyclin B2. Cyclin B1 proved to be an essential gene; no homozygous B1-null pups were born. In contrast, nullizygous B2 mice developed normally and did not display any obvious abnormalities. Both male and female cyclin B2-null mice were fertile, which was unexpected in view of the high levels and distinct patterns of expression of cyclin B2 during spermatogenesis. We show that the expression of cyclin B1 overlaps the expression of cyclin B2 in the mature testis, but not vice versa. Cyclin B1 can be found both on intracellular membranes and free in the cytoplasm, in contrast to cyclin B2, which is membrane-associated. These observations suggest that cyclin B1 may compensate for the loss of cyclin B2 in the mutant mice, and implies that cyclin B1 is capable of targeting the p34 cdc2 kinase to the essential substrates of cyclin B2.

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define ‘S-phase promoting factor’ (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

Products currently available for monitoring children\'s email usage are either considered to encourage dubious ethical behaviour or are time consuming for parents to administer. This paper describes the development of a new email client application for children called Mifrenz. This new application gives parents the ability to let their children safely use email, with the minimum of intervention. It was developed using mostly free software and also with the desire to provide real first hand programming examples to demonstrate to students.

The cyclins comprise a family of proteins which combine with protein kinase subunits encoded by members of the cdc2 family. The active protein kinase thus formed phosphorylates target proteins in the cell and promotes certain cell cycle transitions. Cyclins A and B show the unusual property of sudden and specific proteolysis shortly before the metaphase-anaphase transition during mitosis. The destruction of the cyclins leads to rapid loss of activity of their kinase companions. The 'simple idea' referred to in the title of this article was that accumulation of cyclin formed maturation promoting factor (MPF), the enzyme that promotes the G2-M transition, and cyclin destruction turned off MPF. The complexity refers to additional controls of cdc2 activity, such as its reversible phosphorylation. Furthermore, many new members of the cyclin family have recently been discovered that may play a role in the regulation of the G1-S transition, and in higher organisms, the cdc2 family is also more numerous than was at first appreciated. Cyclins make important contributions both to subcellular localization and the substrate specificity of their companion kinase subunits. It is too early to say if the entire range of cyclin-like and cdc2-like protein kinases are involved in the control of the cell cycle.

All types of double-stranded RNA (DSRNA) tested inhibit protein synthesis in rabbit reticulocyte lysates. The inhibition is characterized by its strongly biphasic kinetics, and can be enhanced by preincubation of the lysate with dsRNA in the absence of protein synthesis. Only properly and extensively matched dsRNA (greater than about 50 base pairs) has this property; no form of DNA, single-stranded RNA or even RNA-DNA hybrids act as inhibitors in this way. The cause of the inhibition appears to be a failure of initiator tRNA to associate with native ribosomal subunits in the initiation process (Darnbrough, C., Hunt, T., and Jackson, R. J. (1973) Biochem. Biophys. Res. Commun. 48, 1556-1564). We have shown that this block is not accompanied by stable association of dsRNA with the ribosomes. There are several reasons to believe that the mechanism of action of dsRNA may be complex with the possible involvement of at least one catalytic step. First, the lysate is inhibited by levels of dsRNA at which ribosomes are present in 100-fold excess over base pairs of dsRNA present. Second, high concentrations of dsRNA (greater than 10 mug per ml) are not inhibitory, but can in some, but not all experiments, reverse the inhibition caused by lower levels of dsRNA. Third, a lysate which has been inhibited by dsRNA, when mixed with a fresh lysate will inhibit synthesis in the mixture much more severely than would be expected from the concentration of dsRNA now present. These results indicate that low levels of dsRNA promote the formation of an inhibitor which may exist in two forms: one that is reversible by high levels of dsRNA and one that is irreversible.

The cyclins are a family of proteins encoded by maternal mRNA. Cyclin polypeptides accumulate during interphase and are destroyed during mitosis at about the time of entry into anaphase. We show here that Xenopus oocytes contain mRNAs encoding two cyclins that are major translation products in a cell-free extract from activated eggs. Cutting these mRNAs with antisense oligonucleotides and endogenous RNAase H blocks entry into mitosis in a cell-free egg extract. The extracts can enter mitosis if either of the cyclin mRNAs is left intact. We conclude that the synthesis of these cyclins is necessary for mitotic cell cycles in cleaving Xenopus embryos.

Summary Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they control important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p107. We now show, using biologically active fusion proteins, that cyclin A can direct the binding of the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p107, as well as activate its histone H1 kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin A-p33cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an activating and targeting subunit of p33cdk2 The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progression and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.

This paper describes the design and implementation of a method for allowing the user interface of a software application to be translated by the end user into any other language. It is proposed that if used by the software industry this technique will increase the availability of software to minority groups. The research involved the modification of an existing email application for children (www.mifrenz.com) by providing a tool for parents to modify the pre-installed translations created using automated translation tools. Standard software internationalisation techniques available with modern programming languages were extensively used. This work resulted in a fully implemented product that has been sold in 11 countries and has confirmed usage in Dutch, French, German, Spanish, Norwegian, Russian, Swedish, and English. It is concluded that with the advent of automated translation tools and giving the end user the ability to modify the translation, as described in this paper, means that it is now possible for all software to be delivered with any interface language at a minimal cost.

Oogenesis. Developmental Biology: A Comprehensive Synthesis, Vol. 1. Edited by Leon W. Browder. Plenum: 1985. Pp.632. $75, £71.25.