Functional importance of conserved domains in the flowering-time gene CONSTANS demonstrated by analysis of mutant alleles and transgenic plants.

CONSTANS promotes flowering of Arabidopsis in response to long-day conditions. We show that CONSTANS is a member of an Arabidopsis gene family that comprises 16 other members. The CO-Like proteins encoded by these genes contain two segments of homology: a zinc finger containing region near their amino terminus and a CCT (CO, CO-Like, TOC1) domain near their carboxy terminus. Analysis of seven classical co mutant alleles demonstrated that the mutations all occur within either the zinc finger region or the CCT domain, confirming that the two regions of homology are important for CO function. The zinc fingers are most similar to those of B-boxes, which act as protein-protein interaction domains in several transcription factors described in animals. Segments of CO protein containing the CCT domain localize GFP to the nucleus, but one mutation that affects the CCT domain delays flowering without affecting the nuclear localization function, suggesting that this domain has additional functions. All eight co alleles, including one recovered by pollen irradiation in which DNA encoding both B-boxes is deleted, are shown to be semidominant. This dominance appears to be largely due to a reduction in CO dosage in the heterozygous plants. However, some alleles may also actively delay flowering, because overexpression from the CaMV 35S promoter of the co-3 allele, that has a mutation in the second B-box, delayed flowering of wild-type plants. The significance of these observations for the role of CO in the control of flowering time is discussed.

NDT80 and the meiotic recombination checkpoint regulate expression of middle sporulation-specific genes in Saccharomyces cerevisiae.

Distinct classes of sporulation-specific genes are sequentially expressed during the process of spore formation in Saccharomyces cerevisiae. The transition from expression of early meiotic genes to expression of middle sporulation-specific genes occurs at about the time that cells exit from pachytene and form the meiosis I spindle. To identify genes encoding potential regulators of middle sporulation-specific gene expression, we screened for mutants that expressed early meiotic genes but failed to express middle sporulation-specific genes. We identified mutant alleles of RPD3, SIN3, and NDT80 in this screen. Rpd3p, a histone deacetylase, and Sin3p are global modulators of gene expression. Ndt80p promotes entry into the meiotic divisions. We found that entry into the meiotic divisions was not required for activation of middle sporulation genes; these genes were efficiently expressed in a clb1 clb3 clb4 strain, which fails to enter the meiotic divisions due to reduced Clb-dependent activation of Cdc28p kinase. In contrast, middle sporulation genes were not expressed in a dmc1 strain, which fails to enter the meiotic divisions because a defect in meiotic recombination leads to a RAD17-dependent checkpoint arrest. Expression of middle sporulation genes, as well as entry into the meiotic divisions, was restored to a dmc1 strain by mutation of RAD17. Our studies also revealed that NDT80 was a temporally distinct, pre-middle sporulation gene and that its expression was reduced, but not abolished, on mutation of DMC1, RPD3, SIN3, or NDT80 itself. In summary, our data indicate that Ndt80p is required for expression of middle sporulation genes and that the activity of Ndt80p is controlled by the meiotic recombination checkpoint. Thus, middle genes are expressed only on completion of meiotic recombination and subsequent generation of an active form of Ndt80p.

An Ssn6-Tup1-dependent negative regulatory element controls sporulation-specific expression of DIT1 and DIT2 in Saccharomyces cerevisiae.

Sporulation of the yeast Saccharomyces cerevisiae is a process of cellular differentiation that occurs in MATa/MAT alpha diploid cells in response to starvation. The sporulation-specific genes DIT1 and DIT2, which are required for spore wall formation, are activated midway through the sporulation program, with maximal transcript accumulation occurring at the time of prospore enclosure. In this study, we have identified a negative regulatory element, termed NREDIT, that is located between the start sites of transcription of these divergently transcribed genes. This element, which prevents expression of the DIT1 and DIT2 genes during vegetative growth, reduces expression of a CYC1-lacZ reporter gene more than 1,000-fold and acts in an orientation- and position-independent manner. We found that the ability of NREDIT to turn of expression of the reporter gene and the chromosomal DIT1 and DIT2 genes in vegetative cells requires the Ssn6-Tup1 repression complex. Interestingly, NREDIT-mediated repression of the reporter gene is maintained during sporulation. Derepression during sporulation requires complex interactions among several cis-acting elements. These are present on an approximately 350-bp DNA fragment extending from NREDIT to the TATA box and an approximately 125-bp fragment spanning the TATA box of DIT1. Additionally, a region of NREDIT which is very similar in sequence to UASSPS4, an element that activates gene expression midway through sporulation, contributes both to vegetative repression and to sporulation-specific induction of DIT1. We propose a model to explain the requirement for multiple elements in overcoming NREDIT-mediated repression during sporulation.

A 15-base-pair element activates the SPS4 gene midway through sporulation in Saccharomyces cerevisiae.

Sporulation of the yeast Saccharomyces cerevisiae represents a simple developmental process in which the events of meiosis and spore wall formation are accompanied by the sequential activation of temporally distinct classes of genes. In this study, we have examined expression of the SPS4 gene, which belongs to a group of genes that is activated midway through sporulation. We mapped the upstream boundary of the regulatory region of SPS4 by monitoring the effect of sequential deletions of 5'-flanking sequence on expression of plasmid-borne versions of SPS4 introduced into a MATa/MAT alpha delta sps4/delta sps4 strain. This analysis indicated that the 5' boundary of the regulatory region was within 50 bp of the putative TATA box of the gene. By testing various oligonucleotides that spanned this boundary and the downstream sequence for their ability to activate expression of a heterologous promoter, we found that a 15-bp sequence sufficed to act as a sporulation-specific upstream activation sequence. This 15-bp fragment, designated UASSPS4, activated expression of a CYC1-lacZ reporter gene midway through sporulation and was equally active in both orientations. Extending the UAS fragment to include the adjacent 14-bp enhanced its activity 10-fold. We show that expression of SPS4 is regulated in a manner distinct from that of early meiotic genes: mutation of UME6 did not lead to vegetative expression of SPS4, and sporulation-specific expression was delayed by mutation of IME2. In vivo and in vitro assays suggested that a factor present in vegetative cells bind to the UASSPS4 element. We speculate that during sporulation this factor is modified to serve as an activator of the SPS4 gene or, alternatively, that it recruits an activator to the promoter.

Pulmonary uptake of liposome-associated alpha-tocopherol following intratracheal instillation in rats.

This study examined the uptake and subcellular distribution of alpha-tocopherol in the lung following intratracheal instillation of liposome-associated alpha-tocopherol in rats. The liposomal suspension was composed of dipalmitoylphosphatidylcholine (DPPC) and alpha-tocopherol (molar ratio 7:3), labelled with [3H]alpha-tocopherol and [14C]cholesterol. Following intratracheal administration of the liposomal preparation (2 mg alpha-tocopherol/animal), the recovery of [3H]alpha-tocopherol in the lung was maximal (87% of initial dose) 1 h after treatment; thereafter, alpha-tocopherol levels remained relatively high (no less than 73% of initial dose) for the rest of the 72-h experimental period. This treatment effect/resulted in a 16-fold increase in pulmonary total alpha-tocopherol concentration 72 h post-instillation. No radioactivity was detected in the blood, liver, kidney, pancreas, spleen and heart of animals during the 72-h experimental period. [3H]alpha-Tocopherol was recovered largely from cytosolic (45%) and nuclear (36%) fractions of lung and to a lesser extent, from microsomal (11%) and mitochondrial (9%) fractions. Chromatographic analysis of the subcellular fractions revealed that [3H]alpha-tocopherol was co-eluted with 14C-labelled liposomal lipids. Our in-vitro study, involving the incubation of Fe(3+)-ADP (a pro-oxidant) with mitochondrial or microsomal fractions isolated from lung tissues of animals treated with liposome-associated alpha-tocopherol, provided evidence that alpha-tocopherol levels present in the membranes of these subcellular fractions were sufficient to protect against oxidant-induced lipid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Protective effect of liposome-associated alpha-tocopherol against paraquat-induced acute lung toxicity.

The present study was undertaken to investigate whether alpha-tocopherol, entrapped in liposomes and delivered directly to the lung, could protect against paraquat-induced lung damage in the rat. Plain liposomes (composed of dipalmitoylphosphatidylcholine, DPPC) or DPPC/alpha-tocopherol liposomes were administered intratracheally to animals 24 hr prior to an intraperitoneal injection of paraquat (20 mg/kg); rats were killed 24 or 48 hr after paraquat treatment. Results of this study showed that lungs of animals treated with paraquat were damaged extensively as evidenced by an increase in lung weight and a significant reduction in lung angiotensin-converting enzyme (ACE) activity and cytochrome P450 concentration. Furthermore, paraquat treatment resulted in a significant decrease in reduced glutathione (GSH) concentrations and a marked elevation in microsomal lipid peroxidation levels as measured by the formation of diene conjugates. Pretreatment of rats with DPPC liposomes alone did not alter significantly the paraquat-induced changes of all parameters examined. On the other hand, pretreatment of rats with DPPC/alpha-tocopherol liposomes 24 hr prior to paraquat challenge resulted in a significant increase in pulmonary alpha-tocopherol concentrations and antagonized paraquat-induced changes in lipid peroxidation, GSH/GSSG ratio, lung ACE activity and cytochrome P450 concentrations. Results of this study suggested that alpha-tocopherol, delivered directly to the lung in a liposomal formulation 24 hr prior to paraquat administration, confers protection against paraquat-induced lung damage.