RAD6+ gene of Saccharomyces cerevisiae codes for two mutationally separable deoxyribonucleic acid repair functions.

The response of two mutant alleles of the RAD6+ gene of Saccharomyces cerevisiae to the ochre translational suppressor SUQ5 was determined. Both the ultraviolet sensitivity phenotype and the deficiency in ultraviolet-induced mutagenesis phenotype of the rad6-1 allele were suppressed in a [psi+] background. For the rad6-3 allele, only the ultraviolet-sensitivity phenotype was suppressible in a [psi+] background. An SUQ5 rad6-3 [psi+] strain that was examined showed the normal rad6-3 deficiency in ultraviolet-induced mutagenesis. We propose that the RAD6+ gene is divided into two cistrons, RAD6A and RAD6B. RAD6A codes for an activity responsible for the error-prone repair of ultraviolet-induced lesions in deoxyribonucleic acid but is not involved in a cell's resistance to the lethal effects of ultraviolet light. RAD6B codes for an activity essential for error-free repair of potentially lethal mutagenic damage.

Ultraviolet mutagenesis studies of [psi], a cytoplasmic determinant of Saccharomyces cerevisiae.

UV mutagenesis was used to probe the molecular nature of [psi], a nonmitochondrial cytoplasmic determinant of Saccharomyces cerevisiae involved in the control of nonsense suppression. The UV-induced mutation from [psi+] to [psi-] showed characteristics of forward nuclear gene mutation in terms of frequency, induction kinetics, occurrence of whole and sectored mutant clones and the effect of the stage in the growth cycle on mutation frequency. The involvement of pyrimidine dimers in the premutational lesion giving the [psi-] mutation was demonstrated by photoreactivation. UV-induced damage to the [psi] genetic determinant was shown to be repaired by nuclear-coded repair enzymes that are responsible for the repair of nuclear DNA damage. UV-induced damage to mitochondrial DNA appeared to be, at least partly, under the control of different repair processes. The evidence obtained suggests that the [psi] determinant is DNA.

Agents that cause a high frequency of genetic change from [psi+] to [psi-] in Saccharomyces cerevisiae.

The [psi] factor of yeast is cytoplasmically inherited. Singh, Helms and Sherman (1979) reported that high concentrations of KCl and of ethylene glycol induce the genetic change from [psi+] to [psi-]. In this study, the following agents have been shown to induce the same genetic change: guanidine hydrochloride at 1 mM, dimethyl sulfoxide at 2.5% v/v and ethanol or methanol at 10% v/v. It is likely that a number of other agents also cause the change, namely 2 M glycerol, M succinate, M glutamate and M MgCl2. Most of these agents induce the change at very high frequencies; with some, the frequency is 100%. Although the observed phenotypic change can also occur as a result of chromosomal gene mutation, no changes of this type were identified. Some of the agents also cause mutation from [rho+] to [rho-] and from killer to sensitive.

Reversion from suppression to nonsuppression in SUQ5 [psi+] strains of yeast: the classificaion of mutations.

Reversion from the suppressed to nonsuppressed phenotype in strains of geno;type SUQ5 [psi+] ade2-1 his5-2 lys1-1 can1-100 ura3-1 has been induced by treatment with ethyl methanesulphonate, nitrosoguanidine or UV (254 nm) light. Spontaneously occurring revertants have also been selected by two different methods. Reversion has been shown to occur through a variety of nuclear mutations and through mutation of [psi+] to [psi-]. Nuclear mutations included back-mutation of SUQ5, antisuppressor mutations that were recessive, semi-dominant or dominant, and dominant or recessive mutations of genes required for the maintenance of the [psi+] factor. Complementation tests by which the various kinds of mutations could be distinguished from one another were designed. The spectra of spontaneously occurring and induced mutations have been described.

The dominant PNM2- mutation which eliminates the psi factor of Saccharomyces cerevisiae is the result of a missense mutation in the SUP35 gene.

The PNM2- mutation of Saccharomyces cerevisiae eliminates the extrachromosomal element psi. PNM2 is closely linked to the omnipotent suppressor gene SUP35 (also previously identified as SUP2, SUF12, SAL3 and GST1). We cloned PNM2- and showed that PNM2 and SUP35 are the same gene. We sequenced the PNM2- mutant allele and found a single G-->A transition within the N-terminal domain of the protein. We tested the effects of various constructs of SUP35 and PNM2- on psi inheritance and on allosuppressor and antisuppressor functions of the gene. We found that the C-terminal domain of SUP35 protein (SUP35p) could be independently expressed; expression produced dominant antisuppression. Disruption of the N-terminal domain of PNM2- destroyed the ability to eliminate psi. These results imply that the domains of SUP35p act in an antagonistic manner: the N-terminal domain decreases chain-termination fidelity, while the C-terminal domain imposes fidelity. Two transcripts were observed for SUP35, a major band at 2.4 kb and a minor band at 1.3 kb; the minor band corresponds to 3' sequences only. We propose a model for the function of SUP35, in which comparative levels of N- and C-terminal domains of SUP35p at the ribosome modulate translation fidelity.

A ribosome-associated inhibitor of in vitro nonsense suppression in [psi-] strains of yeast.

All classes of tRNA-mediated nonsense suppression are much more efficient in yeast cell-free lysates prepared from a [psi+] strain than in those prepared from an isogenic [psi-] strain. Mixed [psi+]/[psi-] lysates do not support efficient suppression. Fractionation of the [psi-] lysate demonstrated the presence of an inhibitor of in vitro suppression that is loosely associated with the 80 S ribosome. The data indicate that the inhibitor is a factor involved in the termination of translation in this simple eukaryote.

Effects of the gonadotrophin-releasing hormone agonist 'Zoladex' upon pituitary and gonadal function in hypogonadal (hpg) male mice: a comparison with normal male and testicular feminized (tfm) mice.

Hypogonadal (hpg) mutant mice, with a congenital deficiency of hypothalamic gonadotrophin-releasing hormone (GnRH), and testicular feminized (tfm) mice, which lack a functional androgen receptor, were used to study the effects of the potent GnRH agonist 'Zoladex' (ICI 118630; D-Ser (Bu(t))6, Azgly10-GnRH) on pituitary and gonadal function. Zoladex (0.5 mg) in a sustained-release lactide-glycolide copolymer depot was administered subcutaneously under anaesthesia and was left in place for 7 days, after which time the effects of the drug upon pituitary and serum gonadotrophin concentrations, glycoprotein hormone subunit mRNAs and testicular morphology were investigated. At the pituitary level, Zoladex treatment resulted in a substantial reduction in LH content in normal males, and LH content was depressed in hpg mice even below the basal levels normally found in these mutants. Pituitary LH content in the Zoladex-treated animals was depressed in the tfm groups, but not to the same levels as those found in the normal and castrated normal mice. Zoladex treatment at the time of castration prevented the post-operative elevation in serum LH associated with castration alone. In the androgen-deficient tfm mouse, Zoladex did not depress the normally elevated serum LH levels. Serum LH in the hpg animals was, in all cases, below the limit of detection of the assay. Pituitary FSH content was depressed into the hpg range in both the normal and castrated animals, but there was no further depression in the hpg mice. The pituitary content was reduced in the tfm mice, again the effects not being as dramatic as in the normal and castrated animals. Serum FSH content, as measured by radioimmunoassay, was depressed by 50% in normal mice; there was no reduction in the hpg mice, however. With regard to pituitary gonadotrophic hormone gene expression, Zoladex administration to normal mice caused a dramatic reduction in LH beta mRNA content, to a level approximating that found in untreated hpg mice. The drug also depressed LH beta mRNA in the castrated group to the hpg range when given at the time of castration, whereas in untreated castrated mice there was a significant increase in LH beta mRNA. In the tfm mouse, which can be considered as a model for long-term failure of androgen feedback, Zoladex again induced a fall in LH beta mRNA, but not to the same extent as in the normal and normal castrated group. Zoladex had no effect on the already low levels of LH beta mRNA found in hpg mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth hormone-deficient dwarfism in the rat: a new mutation.

Mutations in animals have provided insight into many aspects of normal and pathological human physiology. This paper reports the discovery and initial characterization of a new mutant dwarf rat. The mutation, inherited as an autosomal recessive, arose spontaneously in a breeding colony of Lewis rats at the Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, Oxford, U.K., in 1985 and the strain has now been established both in Oxford and at Mill Hill. Body growth in the mutant is retarded such that at 3 months of age both males and females weigh approximately 40% less than their normal litter-mates, and continue to grow at a slower rate. The mutants show a selective reduction in pituitary GH synthesis and storage (pituitary GH concentrations were approximately 10% of normal in males and 6% in females). The concentration of their anterior pituitary trophic hormones (LH, TSH, prolactin and ACTH) were within the normal range in dwarf animals. Exogenous GH treatment for 5 days resulted in an increase in growth rate from 1.5 +/- 0.3 to 3.9 +/- 0.4 g/day in male mutants, and 0.8 +/- 0.2 to 3.1 +/- 0.1 g/day in females. Longitudinal bone growth rates were more than doubled by this treatment from 49 +/- 5 to 100 +/- 10 micron/day in females and from 52 +/- 11 to 131 +/- 16 micron/day in males. Dot blot and Northern blot analysis of pituitary mRNA extracts revealed that the GH message in mutants was between 20 and 25% of normal, and that the GH transcript was of normal size.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of LH injections on testicular steroidogenesis, cholesterol side-chain cleavage P450 mRNA content and Leydig cell morphology in hypogonadal mice.

Hypogonadal (hpg) mice have a congenital deficiency of hypothalamic gonadotrophin-releasing hormone (GnRH) and the gonads consequently lack exposure to gonadotrophins during development. We injected male hpg mice with LH for 10 days to investigate whether LH alone can stimulate normal steroidogenesis in these animals. Control animals had an inactive interstitium and very few germ cells. Testicular content of androgens was undetectable by radioimmunoassay in control animals unless a single injection of LH was given 1 h before death, when androgens were just detectable. Control testes incubated in vitro with [3H]pregnenolone demonstrated that without gonadotrophin stimulation pregnenolone was metabolized only to progesterone in significant amounts. Assay for cholesterol side-chain cleavage cytochrome P450 (P450scc) mRNA showed basal expression in saline-treated hpg mouse testis. LH treatment induced hypertrophy and hyperplasia of Leydig cells and division of germ cells. Testicular androgen content increased significantly, with testosterone and androstenedione as the major androgens. LH-treated testes incubated with [3H]pregnenolone in vitro had a greater synthetic capacity for testosterone, suggesting an increase in 17 alpha-hydroxylase/C17-20-lyase activity. Basal and human chorionic gonadotrophin-stimulated androgen production in vitro increased markedly following LH treatment to levels previously described in the normal adult animal. LH treatment caused a rapid and transient increase in the hybridization of P450scc mRNA which was sevenfold greater than that of saline-treated controls when the animals were killed 1 h after the last injection but fell to control levels within 24 h of cessation of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Ovariectomy or Oestrogen Implants upon Pituitary Function in Female Hypogonadal Mice Bearing Normal Fontal Preoptic Area Grafts.

Abstract The effects of ovariectomy and oestrogen feedback for 10 days upon pituitary and serum luteinizing hormone (LH) content, pituitary glycoprotein subunit messenger ribonucleic acid (mRNA) and prolactin mRNA content in normal females, female hypogonadal mice and hypothalamic grafted female hypogonadal mice, bearing a graft of normal mouse preoptic area tissue into the third ventricle, have been investigated. In normal females ovariectomy resulted in a rise in serum LH, LHbeta-subunit and common alpha-subunit mRNAs with no significant change in pituitary LH content or follicle-stimulating hormone (FSH) beta-subunit mRNA. In the hypogonadal females, preoptic area grafting resulted in an elevation in all of the above parameters into the normal range. Ovariectomy in this group resulted in a further elevation of serum LH, LHbeta-subunit and alpha-subunit mRNAs with no change in pituitary LH content or FSHbeta-subunit mRNA, which in all cases were comparable to ovariectomized normal animals. Oestrogen treatment caused a fall in pituitary LH content and the serum LH fell below the detection of the assay. LHbeta-subunit and a-subunit mRNA mirrored this fall but there was no change in FSHbeta-subunit hybridization. These experiments suggest that even though normal neuronal input to the gonadotrophin-releasing hormone neurons is disrupted, oestrogen-induced negative feedback can still occur in grafted female hypogonadal animals. Gonadotrophin-releasing hormone neurons are reported to lack oestrogen receptors but feedback within this graft by co-transplanting oestrogen-sensitive neurons remains a possibility, as does feedback at the level of the host median eminence where graft axons extend to the pituitary portal vessels. The similarity of the response in normal and grafted animals indicates that these actions of oestrogen may be effected predominantly at the pituitary level.

DNA synthesis in UV-irradiated yeast.

The amount and quality of DNA synthesized in excision-defective strains of yeast was assayed using alkaline sucrose gradients. It was observed that, in such strains, there was less newly synthesized DNA in irradiated cells and that this material was in smaller pieces than in unirradiated controls. The molecular weight was inversely proportional to dose. The low molecular weight DNA chased into normally high molecular weight material during a post-pulse incubation period. This chase was inhibited by higher doses of UV, by hydroxyurea, and required the function of the RAD18 gene. The RAD6 gene function was necessary to prevent degradation of template DNA in irradiated excision-defective strains.

The role of dimer excision in liquid-holding recovery of UV-irradiated haploid yeast.

We have directly tested the theory that liquid-holding recovery is due to an increase in the efficiency of excision-repair during holding in non-growth conditions, by assaying the dimers present in UV-irradiated cells held in saline, in growth medium or first in saline then in growth medium. We observed no differences in the amount of excision in any conditions. By assaying the kinetics of excision and comparing that with the timing of DNA synthesis, we have tested the theory that holding in growth medium allows more repair by extending the time available for it. We found that the observations were more consistent with the onset of DNA synthesis being dependent on the amount of repair rather than the converse. We have analysed the role of repair in liquid-holding recovery in a series of split-dose experiments. As Parry and Parry found, yeast cells which have been irradiated and held in non-growth conditions were much more resistant to further UV-irradiation. The increase in resistance was proportional both to the degree of fractionation of the dose and to the size of the first dose. No effect was observed if this was below 30 J . m-2. We found that the cells were able to excise more of the dimers induced if the UV dose was fractionated. We have shown that part of this increase in efficiency of excision is due to the relief of "dimer interference". "Dimer interference" is the name given to the inhibition of excision of a dimer by the presence of a neighbouring dimer. Most of the increase in efficiency, however, was due to the induction of more efficient excision repair per se, that is the excision of a greater fraction of the dimers present than could be excised in uninduced cells. Among the incidental observations we have made which are new and likely to be of interest are (1) that stationary phase cells showed a lag in the onset of excision, but log phase cells did not; (2) that excision was nevertheless constitutive in that it occurred in the presence of concentrations of cycloheximide inhibitory to protein synthesis and (3) that caffeine affected but did not inhibit dimer excision.

Repair of UV-induced DNA damage and survival in yeast. I. Dimer excision.

The amount of pyrimidine dimer UV photoproduct lost from the DNA of irradiated yeast cells during dark incubation has been measured in various conditions. It was found that no dimers were lost when cells were incubated in saline. When the cells were incubated, with aeration, in a full growth medium, dimers were lost, most excision being complete within 4 h. Not all dimers were lost and the number lost was a function of UV dose. Maximum loss, amounting to 50 000 dimers per genome was observed after 4000 or 6000 erg/mm2 of UV. At higher doses, the number excised declined. Making the assumptions that dimers are the principal lethal product of UV, that a single dimer remaining in its genome is enough to prevent a cell from multiplying and that excision is the principal dark-repair process in yeast, these data were incorporated into the repair term of an expression relating survival to repair8 and it was found that the survival of yeast at doses up to 2000 erg/mm2 of UV could be quite accurately predicted. This is the first time it has been possible to account for survival in terms of measured repair. It is suggested that the divergence of the predicted and observed curves at higher doses is due to other processes known to exist in yeast.

Motivating signage prompts safety belt use among drivers exiting senior communities.

Senior drivers are vulnerable to automobile crashes and subsequent injury and death. Safety belts reduce health risks associated with auto crashes. Therefore, it is important to encourage senior drivers to wear safety belts while driving. Using an AB design, replicated five times, we evaluated the short- and long-term effects of a sign with the message "BUCKLE UP, STAY SAFE" attached to a stop sign at the exits of five different senior communities. Safety belt use was stable during two pretreatment assessments averaged across the five sites and 250 drivers (72% and 68% usage), but significantly increased following installation of these signs (94% usage). Six months after installation of the signs, the effect persisted (88% usage). Use of such signs may be a cost-effective way of promoting safety belt use.