The Kluyveromyces lactis toxin causes an arrest of sensitive yeast cells in the G1 phase of the cell division cycle. Two complementary genetic approaches have been undertaken in the yeast Saccharomyces cerevisiae to understand the mode of action of this toxin. First, two sequences conferring toxin resistance specifically in high copy number have been isolated and shown to encode a tRNA(Glu3) and a novel polypeptide. Disruption of the latter sequence in the yeast genome conferred toxin resistance and revealed that it was nonessential, while the effect of the tRNA(Glu)3 was highly specific and mediated resistance by affecting the toxin's target. An alpha-specific, copy number-independent suppressor of toxin sensitivity was also isolated and identified as MATa, consistent with the observation that diploid cells are partially resistant to the toxin. Second, in a comprehensive screen for toxin-resistant mutants, representatives of 13 complementation groups have been obtained and characterized to determine whether they are altered in the toxin's intracellular target. Of 10 genes found to affect the target process, one (KTI12) was found to encode the novel polypeptide previously identified as a multicopy resistance determinant. Thus, both loss of KTI12 function and elevated KTI12 copy number can cause resistance to the K. lactis toxin.

Polyketide metabolism in S. fradiae is strongly dependent on the thioesterase activity encoded by orf5 (tylO). It is proposed that the TylG complex might operate with a significant error frequency and be prone to blockage with aberrant polyketides. A putative editing activity associated with TylO might be essential to unblock the polyketide synthase complex and thereby promote antibiotic accumulation.

The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.

Context. The quest for the cosmological parameters that describe our universe continues to motivate the scientific community to undertake very large survey initiatives across the electromagnetic spectrum. Over the past two decades, the Chandra and XMM-Newton observatories have supported numerous studies of X-ray-selected clusters of galaxies, active galactic nuclei (AGNs), and the X-ray background. The present paper is the first in a series reporting results of the XXL-XMM survey; it comes at a time when the Planck mission results are being finalised. Aims. We present the XXL Survey, the largest XMM programme totaling some 6.9 Ms to date and involving an international consortium of roughly 100 members. The XXL Survey covers two extragalactic areas of 25 deg 2 each at a point-source sensitivity of ∼5 × 10 −15 erg s −1 cm −2 in the [0.5−2] keV band (completeness limit). The survey's main goals are to provide constraints on the dark energy equation of state from the spacetime distribution of clusters of galaxies and to serve as a pathfinder for future, wide-area X-ray missions. We review science objectives, including cluster studies, AGN evolution, and large-scale structure, that are being conducted with the support of approximately 30 follow-up programmes. Methods. We describe the 542 XMM observations along with the associated multi-λ and numerical simulation programmes. We give a detailed account of the X-ray processing steps and describe innovative tools being developed for the cosmological analysis. Results. The paper provides a thorough evaluation of the X-ray data, including quality controls, photon statistics, exposure and background maps, and sky coverage. Source catalogue construction and multi-λ associations are briefly described. This material will be the basis for the calculation of the cluster and AGN selection functions, critical elements of the cosmological and science analyses. Conclusions. The XXL multi-λ data set will have a unique lasting legacy value for cosmological and extragalactic studies and will serve as a calibration resource for future dark energy studies with clusters and other X-ray selected sources. With the present article, we release the XMM XXL photon and smoothed images along with the corresponding exposure maps.

The Kluyveromyces lactis toxin is a heterotrimeric protein which irreversibly arrests proliferation of sensitive Saccharomyces cerevisiae cells in the G1 phase of the cell cycle. By expressing the gamma subunit of the toxin in sensitive yeast cells from a conditional promoter, it was previously demonstrated that it alone is required for inhibition (Tokunaga et al. (1989). Nucleic Acids Res. 17, 3435-3446). Here we show that, like native exogenous toxin, intracellular gamma subunit expression promotes a striking arrest of sensitive cells in G1. However, unlike the G1 arrest caused by native toxin, that induced by the gamma subunit alone does not result in reduced cellular viability and is fully and rapidly reversible, suggesting that the G1 arrest and the irreversibility of action may reflect different aspects of the toxin's interaction with sensitive cells. We have selected a large number of S. cerevisiae mutants which are highly resistant to the toxin in order to study its mode of action in more detail. Complementation analysis demonstrated that all but one of the mutants were recessive and these defined four separate genes. Members of two complementation groups concurrently acquired resistance to intracellular gamma subunit expression, suggesting that they contain a modified toxin target site. The other two genes appear to be required for entry of the gamma subunit into the sensitive cells since these mutants, while refractory to exogenous toxin, were fully sensitive to intracellular gamma subunit expression.

The response of Saccharomyces cereuisiae cells to the toxin produced by certain strains of Kfuyueromyces factis was studied. The toxin caused an arrest of sensitive cells in the unbudded (Gl) phase of the cell cycle, consistent with the accumulation of cells with an unreplicated (Gl) content of DNA in treated populations. However, toxintreated cells were not proficient for mating. The effects of the toxin were dependent on its continuous presence for over an hour and removal of cells into fresh medium at earlier times prevented inhibition. Following toxin treatment, cells increased in volume and continued to synthesize protein and RNA, suggesting that they were able to continue growth in the absence of division. However, several lines of evidence suggested that the toxin does not simply block proliferation in G1, but that another continuous or post-Gl event is also affected. Possible models to explain these observations are discussed.

The Kluyveromyces lactis toxin is a protein containing three subunits (alpha, beta and gamma) which causes sensitive yeast cells to arrest proliferation in the G1 phase of the cell cycle. Despite the toxin's complex structure, the gamma subunit appears to be the only component required for it to arrest proliferation since intracellular expression of the gamma polypeptide alone in a sensitive yeast strain mimics the effect of the exogenous native toxin. The toxin alpha subunit shows sequence similarity to a variety of chitinases and here we report that the toxin is a potent exochitinase. The exochitinase activity is absolutely required for its biological activity against sensitive Saccharomyces cerevisiae cells and allosamidin, a specific inhibitor of chitinases, abolishes the biological activity of the toxin. However, since the alpha subunit is not required for the G1 arrest induced by the toxin, the chitinase activity of the toxin cannot be directly responsible for the ultimate effect of the toxin and most likely plays a role in the initial interaction of the toxin with sensitive cells.

S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641-651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5'-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.