A de novo originated gene depresses budding yeast mating pathway and is repressed by the protein encoded by its antisense strand

Recent transcription profiling studies have revealed an unexpectedly large proportion of antisense transcripts in eukaryotic genomes. These antisense genes seem to regulate gene expression by interacting with sense genes. Previous studies have focused on the non-coding antisense genes, but the possible regulatory role of the antisense protein is poorly understood. In this study, we found that a protein encoded by the antisense gene ADF1 acts as a transcription suppressor, regulating the expression of sense gene MDF1 in Saccharomyces cerevisiae. Based on the evolutionary, genetic, cytological and biochemical evidence, we show that the protein-coding sense gene MDF1 most likely originated de novo from a previously non-coding sequence and can significantly suppress the mating efficiency of baker's yeast in rich medium by binding MAT alpha 2 and thus promote vegetative growth. These results shed new light on several important issues, including a new sense-antisense interaction mechanism, the de novo origination of a functional gene, and the regulation of yeast mating pathway.

A critical reassessment of the role of mitochondria in tumorigenesis

Background Mitochondrial DNA (mtDNA) is being analyzed by an increasing number of laboratories in order to investigate its potential role as an active marker of tumorigenesis in various types of cancer. Here we question the conclusions drawn in most of these investigations, especially those published in high-rank cancer research journals, under the evidence that a significant number of these medical mtDNA studies are based on obviously flawed sequencing results. Methods and Findings In our analyses, we take a phylogenetic approach and employ thorough database searches, which together have proven successful for detecting erroneous sequences in the fields of human population genetics and forensics. Apart from conceptual problems concerning the interpretation of mtDNA variation in tumorigenesis, in most cases, blocks of seemingly somatic mutations clearly point to contamination or sample mix-up and, therefore, have nothing to do with tumorigenesis. Conclusion The role of mitochondria in tumorigenesis remains unclarified. Our findings of laboratory errors in many contributions would represent only the tip of the iceberg since most published studies do not provide the raw sequence data for inspection, thus hindering a posteriori evaluation of the results. There is no precedent for such a concatenation of errors and misconceptions affecting a whole subfield of medical research.

Yeast基因下游二级结构与多聚腺苷作用信号

形成真核生物mRNA 3^末端的多聚腺苷(poly (A))作用涉及前体mRNA下游的三个元件:效率元件(EE)、定位元件(PE)以及实际的剪切和poly(A)作用位点,实验研究提出了一些EE和PE的碱基序列组成。对180个Yeast基因下游(终止密码子后200个碱基)二级结构进行的详细分析显示,约86%的EE、89%的PE与二级结构中碱基非配对的环(发夹环、膨胀环、内环或多分支环)区或连接单链区有关。这个结果提示,反式因子对EE和PE的识别和作用在一定程度上有赖于EE和PE的二级结构特征。借助mRNA二级结构可以提高对EE和PE位点预测的准确性。

Murine MyaK, a member of a family of yeast YAK1-related genes, is highly expressed in hormonally modulated epithelia in the reproductive system and in the embryonic central nervous system

We have cloned a mouse homologue (designated Myak) of the yeast protein kinase YAK1. The 1210 aa open reading frame contains a putative protein kinase domain, nuclear localization sequences and PEST sequences. Myak appears to be a member of a growing family of YAK1-related genes that include Drosophila and human Minibrain as well as a recently identified rat gene ANPK that encode a steroid hormone receptor interacting protein. RNA blot analysis revealed that Myak is expressed at low levels ubiquitously but at high levels in reproductive tissues, including testis, epididymis, ovary, uterus, and mammary gland, as well as in brain and kidney. In situ hybridization analysis on selected tissues revealed that Myak is particularly abundant in the hormonally modulated epithelia of the epididymis, mammary gland, and uterus, in round spermatids in the testis, and in the corpora lutea in the ovary, Myak is also highly expressed in the aqueduct of the adult brain and in the brain and spinal cord of day 12.5 embryos, Mol. Reprod. Dev. 55:372-378, 2000. (C) 2000 Wiley-Liss, Inc.

Secondary structure of yeast genomic downstream region and polyadenylation signals

Polyadenylation of 3 ' -forming in eukaryote concerns three elements located in precursor mRNA downstream region: efficiency element (EE), position element (PE) and the actual site for cleavage and polyadenylation. Several base sequences of EE and PE have

Transcription rates of yeast genes are influenced by the distribution of introns

A comparative analysis on the intron sequence oligonucleotide usages in two sets of yeast genes with higher and lower transcription frequencies, respectively, has shown that the intron sequence structures of the two sets of genes are different. There are more potential binding sites for transcription factors in the introns of the genes with high transcription frequencies. So it is speculated that introns regulate the transcription of genes. But more evidences are needed to favor this speculation. The detailed comparative analyses on the distribution ( length and position) of introns and exons in the two sets of gene sequences also show that there is an obvious boundary between the lengths of the two sets of introns. There is no boundary between the lengths of the two sets of exons, although the means of their lengths are of discrepancy. The situation of the gene lengths ( length of intron and exon) is similar to exon lengths. As far as the relative position, the introns in two sets of genes all have a bias toward the 5' ends of genes. But as the actual position is considered, more introns in high transcription genes have a tendency to be located toward the 5' ends of genes, some even located at 5'-UTR. These results suggest that the gene transcription rates are related to the length of intron, but not to the lengths of exons and genes sequences. The positions of introns may also influence the transcription rates. The transcriptional regulation of introns may be correlative with the transcriptional regulation of the upstream of genes, or be its continuous action.

Statistical analysis of sequence features of introns with positive transcriptional regulation in yeast genes

A great deal of experimental studies have shown that many introns of eukaryotic genes function as regulators of transcription. However, comprehensive studies of this problem have not yet been conducted. After checking the transcription frequencies of some Saccharomyces cerevisiae (yeast), genes and their introns, a remarkable phenomenon was discovered that generally the introns of the genes with higher transcription frequencies are longer, and the introns of the genes with lower transcription frequencies are shorter. This suggests that the longer introns of genes with higher transcription frequencies may contain some characteristic sequence structures, which could enhance the transcription of genes. Therefore, two sets of introns of yeast genes were chosen for further study. The transcription frequencies of the first set of genes are higher (>30), and those of the second set of genes are lower (less than or equal to10). Some oligonucleotides are detected by statistically comparative analyses of the occurrence frequencies of oligonucleotides (mainly tetranucleotides and pentanucleotides), whose occurrence frequencies in the first set of introns; are significantly higher than those in the second set of introns, and are also significantly higher than those in the exons flanking the introns of the first set. Some of these extracted oligonucleotides are the same as the regulatory elements of transcription revealed by experimental analyses. Besides, the distributions of these extracted oligonucleotides in the two sets of introns and the exons show that the sequence structures of the first set of introns are favorable for transcription of genes.

Detection of potential positive regulatory motifs of transcription in yeast introns by comparative analysis of oligonucleotide frequencies

We conducted a comparative statistical analysis of tetra- through hexanucleotide frequencies in two sets of introns of yeast genes. The first set consisted of introns of genes that have transcription rates higher than 30 mRNAs/h while the second set contained introns of genes whose transcription rates were lower than or equal to 10 mRNAs/h. Some oligonucleotides whose occurrence frequencies in the first set of introns are significantly higher than those in the second set of introns were detected. The frequencies of occurrence of most of these detected oligonucleotides are also significantly higher than those in the exons flanking the introns of the first set. Interestingly some of these detected oligonucleotides are the same as well known "signature" sequences of transcriptional regulatory elements. This could imply the existence of potential positive regulatory motifs of transcription in yeast introns. (C) 2003 Elsevier Ltd. All rights reserved.

Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2.

Ure2p is the protein determinant of the Saccharomyces cerevisiae prion state [URE3]. Constitutive overexpression of the HSP70 family member SSA1 cures cells of [URE3]. Here, we show that Ssa1p increases the lag time of Ure2p fibril formation in vitro in the presence or absence of nucleotide. The presence of the HSP40 co-chaperone Ydj1p has an additive effect on the inhibition of Ure2p fibril formation, whereas the Ydj1p H34Q mutant shows reduced inhibition alone and in combination with Ssa1p. In order to investigate the structural basis of these effects, we constructed and tested an Ssa1p mutant lacking the ATPase domain, as well as a series of C-terminal truncation mutants. The results indicate that Ssa1p can bind to Ure2p and delay fibril formation even in the absence of the ATPase domain, but interaction of Ure2p with the substrate-binding domain is strongly influenced by the C-terminal lid region. Dynamic light scattering, quartz crystal microbalance assays, pull-down assays and kinetic analysis indicate that Ssa1p interacts with both native Ure2p and fibril seeds, and reduces the rate of Ure2p fibril elongation in a concentration-dependent manner. These results provide new insights into the structural and mechanistic basis for inhibition of Ure2p fibril formation by Ssa1p and Ydj1p.

Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2.

Ure2p is the protein determinant of the Saccharomyces cerevisiae prion state [URE3]. Constitutive overexpression of the HSP70 family member SSA1 cures cells of [URE3]. Here, we show that Ssa1p increases the lag time of Ure2p fibril formation in vitro in the presence or absence of nucleotide. The presence of the HSP40 co-chaperone Ydj1p has an additive effect on the inhibition of Ure2p fibril formation, whereas the Ydj1p H34Q mutant shows reduced inhibition alone and in combination with Ssa1p. In order to investigate the structural basis of these effects, we constructed and tested an Ssa1p mutant lacking the ATPase domain, as well as a series of C-terminal truncation mutants. The results indicate that Ssa1p can bind to Ure2p and delay fibril formation even in the absence of the ATPase domain, but interaction of Ure2p with the substrate-binding domain is strongly influenced by the C-terminal lid region. Dynamic light scattering, quartz crystal microbalance assays, pull-down assays and kinetic analysis indicate that Ssa1p interacts with both native Ure2p and fibril seeds, and reduces the rate of Ure2p fibril elongation in a concentration-dependent manner. These results provide new insights into the structural and mechanistic basis for inhibition of Ure2p fibril formation by Ssa1p and Ydj1p.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Deletion of yeast CWP genes enhances cell permeability to genotoxic agents

We have previously reported the development of a novel genotoxic testing system based on the transcriptional response of the yeast RNR3-lacZ reporter gene to DNA damage. This system appears to be more sensitive than other similar tests in microorganisms, and is comparable with the Ames test. In an effort to further enhance detection sensitivity, we examined the effects of altering major cell wall components on cell permeability and subsequent RNR3-lacZ sensitivity to genotoxic agents. Although inactivation of single CWP genes encoding cell wall mannoproteins had little effect, the simultaneous inactivation of both CWP1 and CWP2 had profound effects on the cell wall structure and permeability. Consequently, the RNR3-lacZ detection sensitivity is markedly enhanced, especially to high molecular weight compounds such as 4-nitroquinoline-N-oxide (> sevenfold) and phleomycin (> 13-fold). In contrast, deletion of genes encoding representative membrane components or membrane transporters had minor effects on cell permeability. We conclude that the yeast cell wall mannoproteins constitute the major barrier to environmental genotoxic agents and that their removal will significantly enhance the sensitivity of RNR-lacZ as well as other yeast-based genotoxic tests.

Inhibition of progesterone receptor activity in recombinant yeast by soot from fossil fuel combustion emissions and air particulate materials

Numerous environmental pollutants have been detected for estrogenic activity by interacting with the estrogen receptor, but little information is available about their interactions with the progesterone receptor. In this study, emission samples generated by fossil fuel combustion (FFC) and air particulate material (APM) collected from an urban location near a traffic line in a big city of China were evaluated to interact with the human progesterone receptor (hPR) signaling pathway by examining their ability to interact with the activity of hPR expressed in yeast. The results showed that the soot of a petroleum-fired vehicle possessed the most potent anti-progesteronic activity, that of coal-fired stove and diesel fired agrimotor emissions took the second place, and soot samples of coal-fired heating work and electric power station had lesser progesterone inhibition activity. The anti-progesteronic activity of APM was between that of soot from petroleum-fired vehicle and soot from coal-fired establishments and diesel fired agrimotor. Since there was no other large pollution source near the APM sampling sites, the endocrine disrupters were most likely from vehicle emissions, tire attrition and house heating sources. The correlation analysis showed that a strong relationship existed between estrogenic activity and anti-progesteronic activity in emissions of fossil fuel combustion. The discoveries that some environmental pollutants with estrogenic activity can also inhibit OR activity indicate that further studies are required to investigate potential mechanisms for the reported estrogenic activities of these pollutants. (c) 2005 Elsevier B.V. All rights reserved.

Evaluation of soot particles of biomass fuels with endocrine-modulating activity in yeast-based bioassay

Exposure to indoor air pollution (IAP) from the combustion of biomass fuels is an important cause of morbidity and mortality in developing countries. In the work discussed in this paper we evaluated the endocrine activity of soot particles from biomass fuels by using yeast bioassay. These pollutants could have beta-galactosidase activity with a relative potency (RP) about 10(-7)-10(-9) that of estradiol. Soot particles from wood and straw combustion only partially induced beta-galactosidase activity whereas others produced fully inductive activity in the yeast assay system. These pollutants did not have estrogen antagonist and progesterone agonist activity within the defined concentration range. However, these pollutants require 2-4 orders of magnitude higher IC50 to inhibit the activity of progesterone in a similar dose-response manner to mifepristone. We therefore propose that the endocrine activity of some environmental pollutants may be because of inhibition of the progesterone receptor (hPR). GC-MS results showed that substituted polycyclic aromatic hydrocarbon (PAH) compounds, substituted phenolic compounds and derivatives, aromatic carbonyl compounds, and phytosteroids in these soot particles may be mimicking endogenous hormones.