鲤鱼金属硫蛋白基因启动区功能的研究

以氯霉素乙酰化酶作为报讯基因、利用草鱼肾培养细胞瞬时表达系统,对已克隆的鲤鱼金属硫蛋白基因５’－调节区１．６ｋｂ的序列进行了功能分析。从顺式效应和反式效应研究证明：所克隆的鲤鱼ＭＴ基因５‘－调节区具有典型ＭＴ启动子的特性实验发现哺乳动物病毒ＳＶ４０增强子要以加强鱼类ＭＴ启动子的活性,提示在系统进化上鱼类基因不但存在增强子元件,并具有哺乳动物增强子相似的作用方式,而且作用于增强子的反式效应因子也存在     

Modulation of firefly luciferase stability and impact on studies of gene regulation

Two of the reporter enzymes most commonly used in studies of eukaryotic gene expression are chloramphenicol acetyl-transferase (CAT) and firefly luciferase (Luc). CAT has a half-life of about 50 h in mammalian cells, making it useful for transient transfection assays but less suitable for assays with stable cell lines. Luc has a half-life of only 3 h in mammalian cells, making it much more responsive in stable cell lines. Luc instability arises from its sensitivity to proteolysis both in vivo and in vitro. Compounds that resemble its natural substrate, luciferin, act as effective competitive inhibitors in vitro. When these compounds (e.g., phenylbenzothiazole) are added to either prokaryotic or eukaryotic cells, more than tenfold increases in Luc activity can be observed. This increased activity results from a lower rate of degradation of the enzyme in vivo and can be mimicked in vitro as phenylbenzothiazole protects Luc from trypsin digestion while it has no effect on the rate of digestion of alkaline phosphatase.     

Vargula hilgendorfii luciferase: a secreted reporter enzyme for monitoring gene expression in mammalian cells

The small marine ostracod crustacean, Vargula hilgendorfii, produces a bright blue luminous secretion which is ejected into seawater. The luminescence is due to a simple enzyme-catalyzed reaction involving only luciferase, luciferin (substrate), and molecular oxygen. Thus, V. hilgendorfii luciferase (VL) should be useful as a reporter enzyme in studies of gene expression in mammalian cells. Expression plasmids consisting of VL cDNA (vl) linked to the promoters simian virus 40 early region, Rous sarcoma virus long terminal repeat, human elongation factor, or mouse granulocyte colony-stimulating factor were introduced into a series of mammalian cell lines. Following transfection, VL activities in cell extracts and culture media were determined by a rapid light emission assay with V. hilgendorfii luciferin. Parallel experiments were carried out with the chloramphenicol acetyltransferase (CAT)-encoding gene. In all cell lines tested, VL was secreted, allowing the reporter activity to be determined directly from a small aliquot of the culture medium. The results indicate that the secreted VL enzyme is superior to CAT, firefly luciferase, and bacterial luciferase as a convenient and versatile indicator of gene expression in mammalian cells.     

人SCF基因5′旁侧-1190～- 853 AT富集区功能研究

 已有的报告基因和EMSA实验研究表明 ,人干细胞生长因子 (SCF)基因 5′旁侧AT富集区- 1 1 90～ - 853在HeLa和MCF 7细胞中均能增强下游基因转录 ,可能为一个核基质结合区(MAR) ,对人SCF基因的转录发挥调控作用 .为进一步研究该AT富集区的功能 ,将人SCF基因 5′旁侧 - 1 1 90～ - 853AT富集区分别克隆入SV40或CMV启动子前后紧接着CAT报告基因 ,瞬时转染Jurkat,HepG2和 3T3细胞 ,检测CAT报告基因的瞬时表达活性 .结果表明 :人SCF基因 5′旁侧- 1 1 90～ - 853AT富集区在Jurkat和HepG2细胞中 ,对分别由SV40和CMV启动子引导的CAT基因表达均有抑制作用 ;但在 3T3细胞中对SV40启动子的转录活性表现出增强作用 ,对CMV启动子的转录活性无明显影响 .这些结果提示 ,人SCF基因 5′旁侧 - 1 1 90～ - 853AT富集区转录调控具有组织细胞特异性 ,在不同的细胞中可能发挥转录增强或抑制作用     

Activity of the simian virus 40 early promoter-enhancer in herpes simplex virus type 1 vectors is dependent on its position, the infected cell type, and the presence of Vmw175.

We have studied some of the parameters governing the expression of a foreign promoter-reporter gene construct incorporated into herpes simplex virus (HSV) type 1. These include the genetic background of the parental virus, the site of transgene insertion within the HSV genome, and the infected cell type. The genetic background of the vector constructs denoted delta 3 was an HSV type 1 mutant deleted for nearly the entire coding portion of Vmw175 (ICP4), the product of the essential immediate-early gene IE3. For vectors denoted +, the IE3 deletion had been repaired by marker rescue. We used as a reporter gene the bacterial chloramphenicol acetyltransferase (CAT) gene, driven by the simian virus 40 (SV40) early promoter and enhancer region. The SV40-cat hybrid gene was inserted either into the HSV thymidine kinase (TK) locus to create the vectors TKScat delta 3 and TKScat+ or into an intergenic site within the BamHI z fragment of the short unique portion of the viral genome to create the vectors GScat delta 3 and GScat+. In Vero and BHK cells infected with TKScat delta 3, CAT activity was first detected at 10 h postinfection and continued to accumulate until 36 h postinfection. In cells of primate origin infected with the replication-competent vector TKScat+, or in primate cells which complement the IE3 deficiency and which were infected with TKScat delta 3, CAT activity was significantly lower than in cells of rodent origin. However, levels of CAT were increased in the presence of cycloheximide, suggesting that the low production of CAT in primate cells was due to repression of SV40-cat hybrid gene expression. In contrast with results with TKScat delta 3 and TKScat+, CAT activity was not detectable in any of the tested cell types infected with GScat delta 3 or GScat+ except under conditions of cycloheximide reversal. These results show that while HSV gene products expressed in the presence of Vmw175 inhibited SV40-cat expression in the tk locus in a cell-type-specific manner, HSV gene products expressed in the presence or absence of Vmw175 inhibited SV40-cat expression in the BamHI z locus independently of cell type.     

Transformation of primary rat kidney cells by fragments of simian virus 40 DNA.

Linear simian virus 40 (SV40) DNA molecules of genome length and DNA fragments smaller than genome length when prepared with restriction endonucleases and tested for transforming activity on primary cultures of baby rat kidney cells. The linear molecules of genome length (prepared with endonucleases R-EcoRI, R-BamHI, and R-HpaII or R-HapII), a 74% fragment (EcoRI/HpaII or HapII-A), and a 59% fragment (BamHI/HapII-A) could all transform rat kidney cells with the same efficiency as circular SV40 DNA. All transformed lines tested contained the SV40-specific T-antigen in 90 to 100% of the cells, which was taken as evidence that the transformation was SV40 specific. The DNA fragments with transforming activity contained the entire early region of SV40 DNA. Endo R-HpaI, which introduced one break in the early region, apparently inactivated the transforming capacity of SV40 DNA, since no transformation was observed with any of the three HpaI fragments tested. Attempts were made to rescue infectious virus from some of the transformed lines by fusion with permissive BSC-1 cells. Infectious virus was only recovered from the cells transformed by circular form I DNA. No infectious virus could be isolated from any of the other types of transformed cells.     

Limitation in use of heterologous reporter genes for gene promoter analysis. Silencer activity associated with the cloramphenicol acetyltransferase reporter gene

Various heterologous reporter genes have been widely used for the functional characterization of gene promoters. Many such studies often found weak to very strong silencer activities to be associated with specific parts of the basal promoter or further upstream regions. In this study, we carried out a systematic study on human blood coagulation factor IX (hFIX) and anti-coagulant protein C (hPC) genes, previously shown to have silencer activities associated with their 5'-flanking regions containing promoter sequences. With newly constructed chloramphenicol acetyltransferase (CAT) reporter vectors carrying hFIX or hPC gene promoter sequences, we confirmed the strong silencer activities associated with the regions nt -1895 through nt -416 of the hFIX gene or with the region nt -802 through nt -82 of the hPC gene. However, no such silencer activities associated with the specific regions were found when autologous hFIX cDNA, hFIX minigenes, or hPC minigenes were used as reporters in the expression vector system. Relative levels of CAT, hFIX, and hPC proteins produced in the transient assays correlated well with their mRNA levels. Human FIX minigene constructs containing a simian virus 40 (SV40) 3'-untranslated region (UTR) taken from the CAT reporter gene showed no silencer activity, indicating that SV40 3'-UTR sequence of the CAT reporter gene does not contribute to the silencer activity. Expression vectors constructed with the beta-galactosidase gene under the control of hFIX gene promoter sequences also showed no silencer activity associated with the region nt -1895 through nt -416. These findings indicate that silencer activities associated with specific regions of promoter sequences as analyzed with CAT reporter genes may represent artifacts specific to the CAT reporter genes. Our findings strongly suggest a need for re-examination of promoter characterizations of many eukaryotic genes, which have been studied to date with CAT reporter genes.     

Translational control of synthesis of simian virus 40 late proteins from polycistronic 19S late mRNA.

The simian virus 40 (SV40) 19S late mRNA is polycistronic, encoding multiple late proteins: agnoprotein, VP2, and VP3. We constructed a chloramphenicol acetyltransferase (CAT) transient expression vector in which the SV40 sequences between nucleotides 5171 and 1046 (via the SV40 origin of replication and including the late promoter) were inserted 5' to the cat gene; therefore, the AUG for CAT expression occurs after the AUGs for agnoprotein, VP2, and VP3. CAT enzyme activity assayed after transfection of these constructions indicates the level of CAT AUG utilization and, therefore, can be used as a measure of the ability of prior AUGs to intercept scanning ribosomes. Specifically, deletions and point mutations of the viral AUGs resulted in increased CAT enzyme activity owing to increased utilization of the downstream CAT AUG. To compare a variety of mutants, we used the levels of increase to calculate the translational efficiency of the viral AUGs. Some of our data agree with predictions of the modified scanning model (MSM). Little variation in downstream CAT AUG utilization was noted regardless of whether the VP2 AUG (in a weak MSM sequence context) was intact or removed. Hence, a scanning ribosome may easily bypass it. Similar analysis of the VP3 AUG (in a favorable MSM sequence context) demonstrated that it could efficiently intercept ribosomes prior to the downstream AUG. Overall, these data indicate that the structure of the 19S late mRNA and the relative efficiency of translational start codon utilization can account for the VP3/VP2 ratio found in infected cells. The agnoprotein reading frame, depending on how the mRNA precursor is spliced, is either not contained in the mRNA or is terminated near the VP2 AUG. Under these conditions, the ability of the agnoprotein AUG to block downstream CAT AUG utilization was found to be minimal in our assay. However, we directly tested the blocking ability of the agnoprotein AUG under conditions in which the reading frame terminated well after the CAT AUG. Although the agnoprotein AUG lies in a very good sequence context, this direct analysis showed that it interfered minimally with utilization of the CAT AUG when under the control of the SV40 late promoter. However, expected high levels of interference were regained when the late promoter was replaced with the Rous sarcoma virus long terminal repeat.(ABSTRACT TRUNCATED AT 400 WORDS)     

Infection by bacterial pathogens expressing type III secretion decreases luciferase activity: ramifications for reporter gene studies

Pathogenic microbes influence gene regulation in eukaryotic hosts. Reporter gene studies can define the roles of promoter regulatory sequences. The effect of pathogenic bacteria on reporter genes has not been examined. The aim of this study was to identify which reporter genes are reliable in studies concerning host gene regulation by bacterial pathogens expressing type III secretory systems. Human intestinal epithelial cells, T84, Caco-2 and HT-29, were transfected with plasmids containing luciferase (luc), chloramphenicol acetyltransferase (CAT) or beta-galactosidase (beta-gal) as reporter genes driven by the inducible interleukin-8 (IL-8) or constitutively active simian virus 40 (SV40) promoter. Cells were infected with enteropathogenic E. coli or Salmonella typhimurium, and the reporter activity was assessed. Luc activity significantly decreased following infection, regardless of the promoter. The activity of recombinant luc was nearly ablated by incubation with either EPEC or Salmonella in a cell-free system. Activity was partially preserved by protease inhibitors, and immunoblot analysis showed a decreased amount and molecular weight of recombinant luc, suggesting protein degradation. Neither beta-gal nor CAT activity was altered by infection. Disruption of type III secretion prevented the loss of luc activity. We conclude that CAT or beta-gal, but not luc, can be used as reliable reporter genes to assess the impact of pathogenic microbes, especially those expressing type III secretion on host cell gene regulation.     

Transgenic Xenopus laevis tadpoles: a transient in vivo model system for the manipulation of lens function and lens development.

Rodent gamma-crystallin promoters were recognized as lens-specific promoters in micro-injected Xenopus laevis tadpoles and targeted the expression of the chloramphenicol acetyl transferase (CAT) reporter gene to the tadpole lens. The onset of expression coincided with lens cell formation. The level of expression continued to increase up to 9 days of development (stage 47), stayed at that level till at least day 13 and dropped by only 57% at day 21. In contrast, the level of expression of a non-tissue-specific promoter, the SV40 early promoter, decreased rapidly in the eye during development and was only detectable up to stage 44 (day 5). The stability of the CAT activity in the lens was assessed by delivering a pulse of activity from a heat shock promoter-CAT fusion gene. The half-life of the CAT activity in the eye was the same as that in the tail. The increase in CAT activity in the lens thus depends upon continued activity of the injected gamma-crystallin promoters. Our data demonstrate that mammalian promoters can be used to target gene expression to specific tissues during Xenopus laevis development.     

Quantification of expression of linked cloned genes in a simian virus 40-transformed xeroderma pigmentosum cell line.

We wished to determine whether simian virus 40 (SV40)-transformed xeroderma pigmentosum cells, despite their defective DNA repair, were suitable for DNA-mediated gene transfer experiments with linked genes. Expression of a nonselectable gene (cat, coding for chloramphenicol acetyltransferase [CAT]) linked to a selectable gene (gpt, coding for xanthine-guanine phosphoribosyltransferase [XPRT]) in the plasmid pSV2catSVgpt was quantified after transfection of SV40-transformed xeroderma pigmentosum [XP20s(SV40)] and normal human [GM0637(SV40)] fibroblast cell lines. A novel autoradiographic assay with [3H]xanthine incorporation showed 0.5 to 0.7% phenotypic expression of XPRT in both cell lines. Without selection, transient CAT activity was 20 times greater in the GM0637(SV40) than in the XP20s(SV40) cells, and transient XPRT activity was 5 times greater. Both of these transient activities were increased and equalized in both cell lines by transfection with pRSVcat or pRSVgpt. Genotypic transformation to gpt+ occurred at a frequency of 2 X 10(-4) to 4 X 10(-4) in both cell lines with pSV2catSVgpt. After 2 to 3 months in selective medium, stable expression of the (nonselected) cat gene was found in 11 (92%) of 12 gpt-containing clones derived from GM0637(SV40) cells and in 13 (81%) of 16 gpt-containing clones from XP20s(SV40) cells. However, the levels of CAT activity did not correlate with those of XPRT activity, and both of these activities varied more than 100-fold among different clones. Copies (1 to 4) of the gpt gene were integrated in four clones of the GM0637(SV40) cells having an XPRT activity of 1 to 5 nmol/min per mg, but 5 to 80 copies were integrated in four XP20s(SV40) clones with an XPRT activity of 0.8 to 1.8 nmol/min per mg. This study shows that XP20s(SV40) is as suitable for gene transfer experiments as the normal human line GM0637(SV40).