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.

Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.

Upstream regulatory region of zebrafish lunatic fringe: Isolation and promoter analysis

Lunatic fringe (Lfng), one modulator of Notch signaling, plays an essential part in demarcation of tissues boundaries during animal early development, especially somitogenesis. To characterize the promoter of zebrafish 1fng and generate somite-specific transgenic zebrafish, we isolated the upstream regulatory region of zebrafish 1fng by blast search at the Ensembl genome database (http://www. ensembl.org) and analyzed the promoter activity using green fluorescent protein (GFP) as a reporter. Promoter activity assay in zebrafish shows that the 0.2-kb fragment containing GC-box, CAAT-box, and TATA-box can direct tissue-specific GFP expression, while the 0.4-kb and 1.2-kb fragments with further upstream sequence included drive GFP expression more efficiently. We produced 1fngEGFP-transgenic founders showing somite-specific expression of GFP and consequently generated a hemizygous 1fngEGFP-transgenic line. The eggs from 1fngEGFP-transgenic female zebrafish show strong GFP expression, which is consistent to the reverse-transcription polymerase chain reaction PCR (RT-PCR) detection of 1fng transcripts in the fertilized eggs. This reveals that zebrafish 1fng is a maternal factor existing in matured eggs, suggesting that fish somitogcnesis may be influenced by maternal factors.

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.

Differing estrogen activities in the organic phase of air particulate matter collected during sunny and foggy weather in a Chinese city detected by a recombinant yeast bioassay

Total air suspended particles (PM 100) collected from an urban location near a traffic line in Wuhan, China, were examined for estrogen using a recombinant yeast bioassay. Wuhan, located at the central part of China, is the fourth biggest city in China with 7 million populations. Today, Wuhan has developed into the biggest city and the largest traveling center of central China, becoming one of the important bases of industry, education and research. Wuhan is right at the confluent point of Yangzi River, the third longest river in the world, and its largest distributary Hanjiang, with mountains and more than 100 takes in downtown area. Therefore, by its unique landscape, Wuhan has formed clear four seasons with relatively long winter and summer and short spring and autumn. Foggy weather usually happen in early spring. The yeast line used in this assay stably expresses human estrogen receptor-alpha. Weak but clear estrogenic activities were detected in the organic phase of crude extracts of air particle materials (APM) in both sunny and foggy weather by 0.19-0.79 mug E2/gPM(100) which were statistically significantly elevated relative to the blank control responding from 20% to 50% of the maximum E2 response, and the estrogenic activity was much higher in foggy weather than in sunny weather. The estrogenic activities in the sub-fractions from chromatographic separation of APM sampled in foggy days were also determined. The results indicated that the responses of the fractions were obviously higher than the crude extracts. Since there is no other large pollution source nearby, the estrogenic material was most likely from vehicle emissions, house heating sources and oil fumes of house cooking. The GC/MS analysis of the PM100 collected under foggy weather showed that there were many phenol derivatives, oxy-PAHs and resin acids which have been reported as environmental estrogens. These results of the analysis of estrogenic potency in sunny and foggy weather in a subtropical city of China indicate that further studies are required to investigate the actual risks for the associated health and atmospheric system. (C) 2004 Elsevier Ltd. All rights reserved.

Presence of estrogenic activity from emission of fossil fuel combustion as detected by a recombinant yeast bioassay

Estrogenic activities of emission samples generated by fossil fuel combustion were investigated with human estrogen receptor (ER) recombinant yeast bioassay. The results showed that there were weak but clear estrogenic activities in combustion emissions of fossil fuels including coal, petroleum, and diesel. The estrogenic relative potency (RP) of fossil fuel combustion was the highest in petroleum-fired car, followed by coal-fired stove, diesel-fired agrimotor, coal-fired electric power station. On the other hand, the estrogenic relative inductive efficiency (RIE) was the highest in coal-fired stove and coal-fired electric power station, followed by petroleum-fired car and diesel-fired agrimotor. The estrogenic activities in the sub-fractions from chromatographic separation of emitted materials were also determined. The results indicated that different chemical fractions in these complex systems have different estrogenic potencies. The GC/MS analysis of the emission showed that there were many aromatic carbonyls, big molecular alcohol, PAHs and derivatives, and substituted phenolic compounds and derivatives which have been reported as environmental estrogens. The existence of estrogenic substances in fossil fuel combustion demands further investigation of their potential adverse effects on human and on the ecosystem. The magnitude of pollution due to global usage of fossil fuels makes it imperative to understand the issue of fossil fuel-derived endocrine activities and the associated health risks, particularly the aggregated risks stemmed from exposure to toxicants of multiple sources. (C) 2003 Elsevier Science Ltd. All rights reserved.

Global transposable characteristics in the complete DNA sequence of the yeast

Global transposable characteristics in the complete DNA sequence of the Saccharomyces cevevisiae yeast is determined by using the metric representation and recurrence plot methods. On the basis of the correlation distance of nucleotide strings, 16 chromosome sequences of the yeast, which are divided into 5 groups, display 4 kinds of the fundamental transposable characteristics: a short increasing period, a long increasing quasi-period, a long major value and hardly relevant.

A quick isolation method for mutants with high lipid yield in oleaginous yeast

A novel method has been developed to easily isolate the mutants with high lipid yield after irradiating oleaginous yeast cells with carbon ions of energy of 80 MeV/u. Pre-selection of the mutants after ion irradiation was performed with culture medium in which the concentration of cerulenin, a potent inhibitor of fatty acid synthetase, was at 8.96 mu mol/l. Afterwards, lipid concentration in the fermentation broth of the pre-selected colonies was estimated by the sulfo-phospho-vanillin reaction instead of the conventional methanol-chloroform extraction. Two mutants with high lipid yield have been successfully selected out by the combined method. This easy and simple method is much less time-consuming but very efficient in the mutant isolation, and it has demonstrated great potential on mutation breeding in oleaginous microorganism.

Inactive and mutagenic effects induced by carbon beams of different LET values in a red yeast strain

To evaluate biological action of microorganism exposed to charged particles during the long distance space exploration. Induction of inactivation and mutation in a red yeast strain Rhodotorula glutinis AY 91015 by carbon beams of different LET values (14.9-120 0 keV mu m(-1)) was investigated It was found that survival curves were exponential, and mutation curves were linear for all LET values The dependence of inactivation cross section on LET approached saturation near 120 0 keV mu m(-1) The imitation cross section saturated when LET was higher than 582 keV mu m(-1) Meanwhile, the highest RBEI for inactivation located at 120 0 key mu m(-1) and the highest RBEm for mutation was at 58.2 key mu m(-1) The experiments imply that the most efficient mutagenic part of the depth dose profile of carbon ion is at the plateau region with intermediate LET value in which energy deposited is high enough to Induce mutagenic lesions but too low to induce over kill effect in the yeast cells (C) 2010 Elsevier B V All rights reserved

~(12)C离子束辐照对酵母发酵能力的影响；Improved ethanol fermentation of a yeast mutant by C-12 ion beam irradiation

利用100MeV/u的12C6+离子束辐照酵母Saccharomyces cerevsiea YY,选育出一株高产突变菌株C03A,考察C03A发酵过程中不同温度、pH、糖汁浓度对发酵的影响。通过正交实验确定最佳发酵条件为:糖汁浓度24%、温度35℃、pH5.0。在10L发酵罐实验中,C03A发酵速率相对原始菌株高,36h发酵完全,比原始菌株缩短12h;发酵产酒率达到13.2%(V/V),比原始菌株高1.6%(V/V)。