Haematopoietic stem cell donor registries: World Marrow Donor Association recommendations for evaluation of donor health.

The ability to identify unrelated haematopoietic stem cell donors in one country for recipients in another country requires cooperation and standardization in many areas. The donor assessment and testing are very important issues affecting quality and safety of donation. This special report details the World Marrow Donor Association's recommended procedures regarding the medical evaluation of donors, with the intent to protect the volunteer from the risk to damage his health and to offer the recipient the appropriate quality of stem cells. This document describes criteria for permanent or temporary deferral, guidelines for risk evaluation of infectious disease, examples of conditions requiring assessment and questionnaires designed to elicit relevant information about a donor's medical history and general health.

Informed consent--suggested procedures for informed consent for unrelated haematopoietic stem cell donors at various stages of recruitment, donor evaluation, and donor workup.

The Ethics Working Group of the World Marrow Donor Association (WMDA) was established to address the increasing and complex number of ethical issues surrounding unrelated haematopoietic stem cell donation where the selected donor and recipient reside in different countries. This paper considers the topic of informed donor consent, but recognises that the recommendations contained within the paper may be subject to cultural variances in interpretation, and to adjustment to meet the legal requirements of individual countries. Nevertheless, the extent of international cooperation establishes sufficient common denominators for the recommendations to be widely adhered to in the interests of best practice.

Triplet energies of pi-conjugated polymers.

Using pulse radiolysis and triplet energy transfer has enabled us to measure the triplet energies in a broad range of different pi-conjugated polymers. In all cases we find that the 1 (3)B(u) is of order 0.6 to 1 eV below the 1 (1)B(u), indicative of localized triplet states with strong electron-electron correlation. We also observe that the 1 (1)A(g)-1 (3)B(u) gap decreases linearly as the 1 (1)A(g)-1 (1)B(u) gap decreases even though polymers with very different structure have been studied. This surprising result suggests that polymers with singlet gap <1.3 eV will have a triplet ground state.

Dynamic interaction of DNA damage checkpoint protein Rad53 with chromatin assembly factor Asf1.

The evolutionarily conserved yeast checkpoint protein kinase Rad53 regulates cell cycle progression, transcription, and DNA repair in response to DNA damage. To uncover potential regulatory targets of Rad53, we identified proteins physically associated with it in vivo using protein affinity purification and tandem mass spectrometry. Here we report that Rad53 interacts in a dynamic functional manner with Asf1, a chromatin assembly factor recently shown to mediate deposition of acetylated histones H3 and H4 onto newly replicated DNA. Biochemical and molecular genetic studies suggest that Asf1 is an important target of the Rad53-dependent DNA damage response and that Rad53 may directly regulate chromatin assembly during DNA replication and repair.

Differential toxicities of anticancer agents among DNA repair and checkpoint mutants of Saccharomyces cerevisiae.

Most cytotoxic anticancer agents damage DNA directly, interfere with DNA metabolism or chromosome segregation, and are particularly toxic in dividing cells. Although a considerable amount of information on the mechanisms of action of these agents is available, the molecular bases for selective tumor cell killing by chemotherapy are largely unknown. Many genetic alterations found in sporadic and hereditary cancers affect functions in DNA repair and cell cycle control and result in sensitivity to DNA damaging agents. We have therefore set out to determine the effects of these cancer mutations on sensitivity or resistance to various chemotherapeutic agents. Because most of the affected genes are well conserved among eukaryotes, we have carried out a comprehensive analysis of a panel of isogenic yeast strains, each defective in a particular DNA repair or cell cycle checkpoint function, for sensitivity to the Food and Drug Administration-approved cytotoxic anticancer agents. Widely different toxicity profiles were observed for 23 agents and X-rays, indicating that the type of DNA repair and cell cycle checkpoint mutations in individual tumors could strongly influence the outcome of a particular chemotherapeutic regimen.

From molecular to modular cell biology.

Cellular functions, such as signal transmission, are carried out by 'modules' made up of many species of interacting molecules. Understanding how modules work has depended on combining phenomenological analysis with molecular studies. General principles that govern the structure and behaviour of modules may be discovered with help from synthetic sciences such as engineering and computer science, from stronger interactions between experiment and theory in cell biology, and from an appreciation of evolutionary constraints.

Drug target validation and identification of secondary drug target effects using DNA microarrays.

We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication.

Pre-B cell receptor-mediated selection of pre-B cells synthesizing functional mu heavy chains.

Ig gene rearrangements could generate V(H)-D-J(H) joining sequences that interfere with the correct folding of a mu-chain, and thus, its capability to pair with IgL chains. Surrogate light (SL) chain might be the ideal molecule to test the capacity of a mu-chain to pair with a L chain early in development, in that only pre-B cells that assemble a membrane mu-SL complex would be permitted to expand and further differentiate. We have previously identified two SL chain nonpairing V(H)81X-mu-chains with distinct V(H)-D-J(H) joining regions. Here, we show that one of these V(H)81X-mu-chains does not rescue B cell development in J(H) knock-out mice, because flow cytometric analysis of bone marrow cells from V(H)81X-mu transgenic J(H) knock-out mice revealed normal numbers of pro-B cells, but essentially no pre-B and surface IgM+ B cells. Immunoprecipitation analysis of transfected pre-B and hybridoma lines revealed that the same mu-chain fails to pair not only with SL chain but also with four distinct kappa L chains. These findings demonstrate that early pre-B cells are selected for maturation on the basis of the structure of a mu-chain, in particular its V(H)-D-J(H) joining or CDR3 sequence, and that one mechanism for this selection is the capacity of a mu-chain to assemble with SL chain. Therefore, we propose a new function of SL chain in early B cell development: SL chain is part of a quality control mechanism that tests a mu-chain for its ability to pair with conventional L chains.

Integrating genetic approaches into the discovery of anticancer drugs.

The discovery of anticancer drugs is now driven by the numerous molecular alterations identified in tumor cells over the past decade. To exploit these alterations, it is necessary to understand how they define a molecular context that allows increased sensitivity to particular compounds. Traditional genetic approaches together with the new wealth of genomic information for both human and model organisms open up strategies by which drugs can be profiled for their ability to selectively kill cells in a molecular context that matches those found in tumors. Similarly, it may be possible to identify and validate new targets for drugs that would selectively kill tumor cells with a particular molecular context. This article outlines some of the ways that yeast genetics can be used to streamline anticancer drug discovery.

CDC5 and CKII control adaptation to the yeast DNA damage checkpoint.

A single double-stranded DNA (dsDNA) break will cause yeast cells to arrest in G2/M at the DNA damage checkpoint. If the dsDNA break cannot be repaired, cells will eventually override (that is, adapt to) this checkpoint, even though the damage that elicited the arrest is still present. Here, we report the identification of two adaptation-defective mutants that remain permanently arrested as large-budded cells when faced with an irreparable dsDNA break in a nonessential chromosome. This adaptation-defective phenotype was entirely relieved by deletion of RAD9, a gene required for the G2/M DNA damage checkpoint arrest. We show that one mutation resides in CDC5, which encodes a polo-like kinase, whereas a second, less penetrant, adaptation-defective mutant is affected at the CKB2 locus, which encodes a nonessential specificity subunit of casein kinase II.

Mating in Saccharomyces cerevisiae: the role of the pheromone signal transduction pathway in the chemotropic response to pheromone.

The mating process in yeast has two distinct aspects. One is the induction and activation of proteins required for cell fusion in response to a pheromone signal; the other is chemotropism, i.e., detection of a pheromone gradient and construction of a fusion site available to the signaling cell. To determine whether components of the signal transduction pathway necessary for transcriptional activation also play a role in chemotropism, we examined strains with null mutations in components of the signal transduction pathway for diploid formation, prezygote formation and the chemotropic process of mating partner discrimination when transcription was induced downstream of the mutation. Cells mutant for components of the mitogen-activated protein (MAP) kinase cascade (ste5, ste20, ste11, ste7 or fus3 kss1) formed diploids at a frequency 1% that of the wild-type control, but formed prezygotes as efficiently as the wild-type control and showed good mating partner discrimination, suggesting that the MAP kinase cascade is not essential for chemotropism. In contrast, cells mutant for the receptor (ste2) or the beta or gamma subunit (ste4 and ste18) of the G protein were extremely defective in both diploid and prezygote formation and discriminated poorly between signaling and nonsignaling mating partners, implying that these components are important for chemotropism.

Genetic analysis of default mating behavior in Saccharomyces cerevisiae.

Haploid Saccharomyces cerevisiae cells find each other during conjugation by orienting their growth toward each other along pheromone gradients (chemotropism). However, when their receptors are saturated for pheromone binding, yeast cells must select a mate by executing a default pathway in which they choose a mating partner at random. We previously demonstrated that this default pathway requires the SPA2 gene. In this report we show that the default mating pathway also requires the AXL1, FUS1, FUS2, FUS3, PEA2, RVS161, and BNI1 genes. These genes, including SPA2, are also important for efficient cell fusion during chemotropic mating. Cells containing null mutations in these genes display defects in cell fusion that subtly affect mating efficiency. In addition, we found that the defect in default mating caused by mutations in SPA2 is partially suppressed by multiple copies of two genes, FUS2 and MFA2. These findings uncover a molecular relationship between default mating and cell fusion. Moreover, because axl1 mutants secrete reduced levels of a-factor and are defective at both cell fusion and default mating, these results reveal an important role for a-factor in cell fusion and default mating. We suggest that default mating places a more stringent requirement on some aspects of cell fusion than does chemotropic mating.