香菇顺式调控元件的克隆及其序列分析*

应用顺式调控元件探测载体G221构建了一个香菇Lentinula edodes基因组文库。G221为大肠杆菌-酿酒酵母穿梭载体,含有一个由酵母Cyc1基因基本启动子控制的lacZ标记基因,能以转录增强活性筛选香菇DNA片段。用这个基因组文库转化酵母菌,获得了一批lacZ阳性转化子。对其中表达较强的阳性转化子进行质粒抽提和双酶切鉴定,筛选到50个香菇顺式调控元件DNA片段。对其中部分片段进行了测序,并对其中一个序列进行了序列分析,鉴别了该序列上的几个与转录相关的特征序列。该研究也探讨了利用酵母表达系统克隆香菇顺式调控元件的可行性。     

Isolation and characterization of sporulation-specific promoters in the yeast Saccharomyces cerevisiae

A library of random yeast genomic DNA:lacZ fusions has been constructed using an episomal yeast-Escherichia coli shuttle vector (pCS1). Plasmid pCS1 requires insertion of a promoter and an in frame ATG codon upstream of its resident truncated lacZ gene to regulate expression in yeast. Yeast genomic DNA fragments of 4-6 kb were generated by partial digestion with Sau3A and ligated into the unique BamHI site of plasmid pCS1 to generate a library of 5 x 10(4) individual E. coli transformants. This library was screened to identify promoter-lacZ fusions that were expressed uniquely during sporulation. Of 342 yeast transformants that exhibited beta-galactosidase activity, two were found to express the lacZ gene in a sporulation-specific manner. This paper presents the characterization of two genomic yeast DNA fragments containing promoters that control lacZ expression during the sporulation process. Expression from the promoter present in plasmid pJC18 occurred from 11-21 hours into the sporulation process, while the promoter in plasmid pJC217 was active from 4-14 hours. Staining of nuclear DNA to correlate nuclear morphology with timing of gene expression showed when each of these promoters was active in terms of the morphological stages of sporulation.     

Yeast cytochrome b2 gene: isolation with antibody probes

An efficient technique was used to clone the gene for yeast cytochrome b2, (a nuclear encoded mitochondrial protein) using the expression vector, lambda gt11 (lac 5 nin 5 c1857 S100). This enables the insertion of yeast DNA into the beta-galactosidase structural gene (lacZ) and promotes synthesis of hybrid proteins. Screening of antigen producing clones in the lambda gt11 recombinant genomic library was achieved using antiserum against cytochrome b2 according to Young and Davis (1983) Two recombinants containing part of the gene coding for cytochrome b2 were isolated and characterized as follows: by their expression in Escherichia coli cells, examined by immuno-blotting with antibodies to pure cytochrome b2. by DNA sequence analysis. One recombinant carries a 3 Kb yeast DNA insert which contains the whole nucleotide sequence encoding cytochrome b2 and a few amino acids of the amino terminal presequence.     

The Saccharomyces cerevisiae MGT1 DNA repair methyltransferase gene: its promoter and entire coding sequence, regulation and in vivo biological functions.

We previously cloned a yeast DNA fragment that, when fused with the bacterial lacZ promoter, produced O6-methylguanine DNA repair methyltransferase (MGT1) activity and alkylation resistance in Escherichia coli (Xiao et al., EMBO J. 10,2179). Here we describe the isolation of the entire MGT1 gene and its promoter by sequence directed chromosome integration and walking. The MGT1 promoter was fused to a lacZ reporter gene to study how MGT1 expression is controlled. MGT1 is not induced by alkylating agents, nor is it induced by other DNA damaging agents such as UV light. However, deletion analysis defined an upstream repression sequence, whose removal dramatically increased basal level gene expression. The polypeptide deduced from the complete MGT1 sequence contained 18 more N-terminal amino acids than that previously determined; the role of these 18 amino acids, which harbored a potential nuclear localization signal, was explored. The MGT1 gene was also cloned under the GAL1 promoter, so that MTase levels could be manipulated, and we examined MGT1 function in a MTase deficient yeast strain (mgt1). The extent of resistance to both alkylation-induced mutation and cell killing directly correlated with MTase levels. Finally we show that mgt1 S.cerevisiae has a higher rate of spontaneous mutation than wild type cells, indicating that there is an endogenous source of DNA alkylation damage in these eukaryotic cells and that one of the in vivo roles of MGT1 is to limit spontaneous mutations.     

Paradoxical decrease in mutant frequencies and chromosomal rearrangements in a transgenic lacZ reporter gene in Ku80 null mice deficient in DNA double strand break repair

Repair of DNA double strand breaks (DSB), either by homologous recombination (HR) or nonhomologous end-joining (NHEJ), is essential to maintain genomic stability. To examine the impact of NHEJ deficiency on genomic integrity in Ku80 null (Ku-) mice, the chromosomally integrated shuttle vector pUR288, which includes a lacZ reporter gene, was used to measure mutations in vivo. Unexpectedly, a significant decrease was found in mutant frequencies of Ku- liver (5.04x10(-5)) and brain (4.55x10(-5)) compared to tissues obtained from normal (Ku+) littermates (7.92x10(-5)and 7.30x10(-5), respectively). No significant difference was found in mutant frequencies in spleen from Ku- (7.21x10(-5)) and Ku+ mice (8.16x10(-5)). The determination of the mutant spectrum in lacZ revealed the almost complete absence of chromosomal rearrangements (R) in Ku- tissues (0.5%, 3/616), a notable distinction from Ku+ controls (16.7%, 104/621). These findings suggest that accurate repair of DSB by HR and elimination of cells with unrepaired DNA damage by apoptosis are capable of maintaining genomic stability of the lacZ reporter in Ku- mice.     

Transgene activity following somatic transgenesis in Nile tilapia Oreochromis niloticus

A detailed study was made of the persistence and expression of a plasmid-enclosed reporter gene construct after intramuscular injection into the somatic muscle tissue of juvenile Nile tilapia Oreochromis niloticus and also the effect of injecting a potentially growth-promoting gene construct. The plasmid-enclosed DNA proved stable at the site of injection, lasting in some cases for up to 6 months, and was, at a very low frequency, detected in gonad tissue, indicating occasional substantial movement from the injected muscle site. It was observed that the reporter gene and regulatory sequences were also functional within the somatic cells. In a comparison of expression levels by direct somatic injection, the 1·6 kb tilapia β-actin regulatory sequence (tiβAP) resulted in c. three-fold higher β-galactosidase activity than the 4·7 kb carp β-actin regulatory sequence (cβAP) when spliced to the lacZ gene. The enhancer element near the end of first intron in the tiβAP, when co-injected with tiβAP/lacZ plasmid at a 3:1 ratio, drove significantly higher reporter activity in somatic cells than the tiβAP/lacZ sequence alone. The introduction of a growth-promoting construct, the Nile tilapia growth hormone gene driven by a tiβAP, yielded no detectable growth enhancement.     

Ligand Recognition in Multiallelic Pheromone Receptors from the Basidiomycete Schizophyllum commune Studied in Yeast

The homobasidiomycete Schizophyllum commune encodes a multiallelic pheromone receptor system that distinguishes more than 20 nonself from at least 2 self pheromones. The well-investigated pheromone response system of the yeast Saccharomyces cerevisiae was used to link the FUS1::lacZ reporter system to the heterologous pheromone receptors from S. commune. To investigate yeast G-protein binding, the unchanged heterologous receptor was compared to constructs carrying an exchange of the 3rd cytoplasmatic loop for the Ste2 sequence. A better coupling could be achieved with the altered constructs. In order to examine activation by single pheromones, an artificial peptide based on the sequence of a new putative pheromone gene, bap2(1), in the Balpha2 mating-type locus encoding the shortest pheromone found so far in fungal mating types was used. Thus, we have reassembled the pheromone recognition of the basidiomycete S. commune and constructed a system ideal for specificity analysis in the yeast S. cerevisiae.     

A Transgenic Mouse Model to Study Transsynaptic Regulation of Tyrosine Hydroxylase Gene Expression

Abstract: Previous studies demonstrated that 9 kb of the rat tyrosine hydroxylase (TH) 5' flanking sequence directed appropriate spatiotemporal expression of a lacZ reporter gene to catecholaminergic cells in the CNS of transgenic mice. In the present study, specificity of transgene expression was further extended to demonstrate cell type-specific functional regulation of lacZ expression using manipulations known to alter endogenous TH expression. Alterations in lacZ reporter expression should parallel changes in endogenous TH levels if the DNA elements mediating these functional changes of TH expression in vivo reside within the 9 kb of the TH promoter region. Naris closure induced an activity-dependent decrease of TH expression in dopaminergic periglomerular cells in the olfactory bulb that was paralleled by down-regulation of lacZ expression in the transgenic mice. Densitometry and image analysis were used to quantify lacZ expression following acute reserpine administration (5 mg/kg, s.c.), which up-regulates endogenous TH. At 48 h postinjection, analysis of OD values indicated a significant increase of X-gal staining in the locus coeruleus and ventral tegmental area but not in the substantia nigra or olfactory bulb of reserpine-treated transgenic animals. These data showed that the 9-kb sequence also mediates cell type-specific transsynaptic regulation of reporter gene expression. Analysis of this transgenic animal offers a useful model system to study in vivo regulation of TH gene expression.     

The influence of antisense gene location on target gene suppression in the fission yeast Schizosaccharomyces pombe

A fission yeast model was employed to investigate the influence of antisense gene location on the efficacy of antisense RNA-mediated target gene suppression. Fission yeast transformants were generated that contained the target lacZ gene at a fixed position and a single copy antisense lacZ gene integrated into various genomic locations, including the same locus as the target gene. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus compared with other genomic locations, indicating that target and antisense gene colocalization is not a critical factor for efficient antisense RNA-mediated gene expression in vivo. Instead, increased lacZ downregulation correlated with an increase in antisense dose, with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number.     

A transgene containing lacZ is expressed in primary sensory neurons in zebrafish.

In order to screen for developmentally active chromosomal domains during zebrafish embryogenesis, we generated transgenic fish by microinjecting two different lacZ reporter constructs into fertilized eggs. Transgenic fish were screened among the progeny of injected fish (F0) crossed to non-injected fish. Groups of 15 to 20 progeny of each cross were tested for lacZ expression and/or transmission of injected sequences using PCR and Southern hybridizations. Progeny from 2 of 102 fish injected with supercoiled constructs containing Rous sarcoma virus promoter sequences showed apparently spatially regulated beta-galactosidase (beta-Gal) activity. However, we were not able to detect this reporter construct in DNA from fins of F1 fish. Injections of a linear reporter construct containing mouse heat-shock promoter sequences revealed transmission of injected sequences to F1 progeny in about 6% of cases (8 of 129 fish, tested with PCR). We found one lacZ-expressing line that showed a spatially and temporally restricted expression of lacZ and, therefore, features typical characteristics of "enhancer trap" lines. In this line, lacZ expression starts at 16 hours post-fertilization in trigeminal ganglion cells. At about 24 hours lacZ expression can be detected in trigeminal ganglion neurons and Rohon-Beard neurons, indicating that the development of these two cell types shows common features. The reporter gene has integrated as a single copy. The founder fish was mosaic: 19% of its offspring (3 of 16 tested animals) carried the reporter construct in their fins; about 51% (13 of 27 tested animals) of the progeny of F1 fish were beta-Gal positive indicating full hemizygosity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The replication behavior of Saccharomyces cerevisiae DNA in human cells

We studied the replication of random genomic DNA fragments from Saccharomyces cerevisiae in a long-term assay in human cells. Plasmids carrying large yeast DNA fragments were able to replicate autonomously in human cells. Efficiency of replication of yeast DNA fragments was comparable to that of similarly sized human DNA fragments and better than that of bacterial DNA. This result suggests that yeast genomic DNA contains sequence information needed for replication in human cells. To examine whether DNA replication in human cells would initiate specifically at a yeast origin of replication, we monitored initiation on a plasmid containing the yeast 2-micron autonomously replicating sequence (ARS) in yeast and human cells. We found that while replication initiates at the 2-micron ARS in yeast, it does not preferentially initiate at the ARS in human cells. This result suggests that the sequences that direct site specific replication initiation in yeast do not function in the same way in human cells, which initiate replication at a broader range of sequences.by J.A. Huberman     

酵母PHO2与PHO4蛋白的激活活性的分析及两者的相互作用

ＰＨＯ２与ＰＨＯ４是酵母ＰＨＯ５基因的两个正调控因子,本文发现,ＰＨＯ２与酵母转录因子ＧＡＬ４的ＤＮＡ结合功能域融合后就能激活报道基因ｌａｃＺ的表达,其激活力受高低磷影响,表明ＰＨＯ２蛋白上存在酸性转录激活区。ＰＨＯ２蛋白上酸性氨基酸丰富的２８７－３２６肽段并非ＰＨＯ２的激活区。在ＰＨＯ２蛋白上２３０位Ｓｅｒ处于磷酸化状态２ＰＨＯ２才有激活作用,表明了这一磷酸化位点可能与ＰＨＯ２的转录激活能力有关     

Construction of tandem repeats of DNA fragments by a polymerase chain reaction-based method

We describe a new application of megaprimer polymerase chain reaction (PCR) for constructing a tandemly repeated DNA sequence using the drought responsive element (DRE) from Arabidopsis thaliana as an example. The key feature in the procedure was PCR primers with partial complementarity but differing melting temperatures (T(m)). The reverse primer had a higher T(m), a 3' end complementary to the DRE sequence and a 5' region complementary to the forward primer. The initial cycles of the PCR were conducted at a lower primer annealing temperature to generate products that served as megaprimers in the later cycles conducted at a higher temperature to prevent annealing of the forward primer. The region of overlap between the megaprimers was extended for generating products with a variable copy number (one to four copies) of tandem DRE sequence repeats (71?bp). The PCR product with four tandem repeats (4× DRE) was used as a template to generate tandem repeats with higher copies (copy number large than four) or demonstrated to bind DRE-binding protein in an yeast one-hybrid assay using promotorless reporter genes (HIS and lacZ). This PCR protocol has numerous applications for generating DNA fragments of repeated sequences.     

Nucleosomal location of the STE6 TATA box and Mat alpha 2p-mediated repression.

It has been proposed that yeast MATa cell-specific genes are repressed in MAT alpha cells by the Mat alpha 2p repressor-directed placement of a nucleosome in a position that incorporates the TATA box of the MATa-specific gene close to the nucleosomal pseudodyad. In this study, we address this proposal directly with a series of plasmids designed to place the MATa-specific STE6 TATA box at different locations in a nucleosome and in the internucleosomal linker. These plasmids contain different lengths of synthetic random DNA between the Mat alpha 2p operator and the TATA box of the STE6 promoter, which is located upstream of a lacZ reporter gene in a multicopy plasmid. We show that in MAT alpha cells, a nucleosome is retained in an identical translational frame relative to the Mat alpha 2p operator in all the constructs investigated, irrespective of the sequence of the DNA wrapped onto the histone octamer. This result shows that the nucleosomal organization of the STE6 promoter in MAT alpha cells is not conferred by the sequence of the promoter itself. No expression of the lacZ reporter gene was detectable in MAT alpha cells in any of the constructs, even with the TATA box located in a short internucleosomal linker. These data indicate that repression of MATa-specific genes in MAT alpha cells does not require the precise translational placement of the TATA box close to the nucleosomal pseudodyad; the gene remains repressed when the TATA box is located within the investigated 250-bp region in the organized chromatin domain abutting the Mat alpha 2p operator in MAT alpha cells and may remain repressed with the TATA box located anywhere within this organized repression domain.     

All phox homology (PX) domains from Saccharomyces cerevisiae specifically recognize phosphatidylinositol 3-phosphate

Phox homology (PX) domains are named for a 130-amino acid region of homology shared with part of two components of the phagocyte NADPH oxidase (phox) complex. They are found in proteins involved in vesicular trafficking, protein sorting, and lipid modification. It was recently reported that certain PX domains specifically recognize phosphatidylinositol 3-phosphate (PtdIns-3-P) and drive recruitment of their host proteins to the cytoplasmic leaflet of endosomal and/or vacuolar membranes where this phosphoinositide is enriched. We have analyzed phosphoinositide binding by all 15 PX domains encoded by the Saccharomyces cerevisiae genome. All yeast PX domains specifically recognize PtdIns-3-P in protein-lipid overlay experiments, with just one exception (a significant sequence outlier). In surface plasmon resonance studies, four of the yeast PX domains bind PtdIns-3-P with high (micromolar range) affinity. Although the remaining PX domains specifically recognize PtdIns-3-P, they bind this lipid with only low affinity. Interestingly, many proteins with "low affinity" PX domains are known to form large multimeric complexes, which may increase the overall avidity for membranes. Our results establish that PtdIns-3-P, and not other phosphoinositides, is the target of all PX domains in S. cerevisiae and suggest a role for PX domains in assembly of multiprotein complexes at specific membrane surfaces.