Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.21.1 (DNase)
 7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We investigated gene transfer in abalone via electroporated sperm. The mobility of sperm electroporated either in seawater or in marine invertebrate physiological solution was as good as that of the control group. The fertilization rate reached as high as 94.7-99.6% (93.0-99.7% for the control group) when 200 eggs were fertilized by 10(6) or 10(7) sperm treated with electroporation at 10 kV and 2(7) pulses for six cycles. Moreover, the fertilization rate of sperm electroporated in the presence of foreign DNA (opAFP-2000CAT) ranging from 0.1 to 3.2 micrograms and at voltages ranging from 2 to 10 kV, at 2(7) or 2(11) pulses for six or 12 cycles showed no differences from the control sperm. After DNase digestion, the genome of the electroporated sperm was analysed by polymerase chain reaction, and it was shown that a 138-bp product was amplified, corresponding to the transgene's amplification product. Southern blotting also showed that a positive band located at the same position as that of opAFP-2000CAT was found in the electroporated sperm after DNase treatment. Analysis by PCR of the genome isolated from a trochophore-stage abalone larva, derived from sperm electroporated with 3.2 micrograms opAFP-2000CAT, showed the existence of foreign DNA in 13 out of 20 examined samples (65%). The integration of the transferred DNA into the genome of transgenic abalone was also shown by Southern blot analysis. Furthermore, CAT activity was positive for the experimental larvae, but the level of CAT expression was lower than that of larvae derived from sperm electroporated with pCAT-Control vector, driven by SV40 promoter and enhancer sequences. These results demonstrate the potential for the use of sperm as mass gene transfer strategy in marine mollusks such as abalone.
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PMID:Sperm as a carrier to introduce an exogenous DNA fragment into the oocyte of Japanese abalone (Haliotis divorsicolor suportexta). 903 81

The rat UDP glucuronosyltransferase UGT2B1 is expressed mainly in the liver where it glucuronidates steroids and environmental toxins and carcinogens. A region between -42 and -55 bp upstream from the UGT2B1 gene transcription start site was previously identified as sharing sequence similarity with the hepatocyte nuclear factor 1 (HNF1) consensus binding site. In this study, the importance of this region in the regulation of the UGT2B1 gene was confirmed by functional and DNA binding assays. A minimal UGT2B1 gene promoter containing the putative HNF1 binding site was fused to the CAT reporter gene and transfected into HepG2 cells. Only low levels of CAT activity were detected. This activity was increased 50-fold when an HNF1 alpha expression vector was co-transfected with the UGT2B1 promoter CAT construct but was not altered when a HNF1 beta expression vector was used. A UGT2B1 promoter construct with the HNF1-like region deleted was not activated by either co-transfected HNF1 expression vector. DNase 1 footprinting and gel-shift analysis demonstrated that nuclear proteins present in both HepG2 cells and rat liver bind to the HNF1-like element. The presence of HNF1 alpha in these nuclear proteins that bind to the HNF1-like element was confirmed by supershift analysis with antisera to HNF1 alpha. Specific binding of nuclear proteins to the HNF1-like element was not seen in extracts from three cell lines derived from nonhepatic tissues. These data strongly suggest that the liver-enriched factor HNF1 alpha binds to, and activates, the UGT2B1 gene promoter
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PMID:HNF1 alpha activates the rat UDP glucuronosyltransferase UGT2B1 gene promoter. 905 41

The insulin receptor gene is induced 8 to 10-fold during adipocyte differentiation. Plasmids containing the promoter, exon 1 and a portion of the first intron from either the mouse or human gene are able to modulate the expression of an insulin receptor/CAT gene 3 to 7-fold during differentiation. We have shown that several nuclear proteins from both preadipocyte and adipocyte nuclear extracts bind to two discrete sites within a 278-bp region in the 5' end of the first intron. Sequence comparison between the first intron of the human gene and the mouse gene shows two regions of sequence identity which correspond to the protein binding regions detected by DNase footprinting. One of these sites binds proteins that are enriched in adipocyte nuclear extracts and can be competed by adipose regulatory element, ARE6.
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PMID:A conserved region in the first intron of the insulin receptor gene binds nuclear proteins during adipocyte differentiation. 939 30

Expression of the lactoferrin gene in a variety of tissues is regulated differentially. We have previously demonstrated that the lactoferrin gene is regulated by estrogen and mitogen in mouse uterus. The mouse lactoferrin gene responded to forskolin, cAMP, TPA and EGF stimulation via two adjacent enhancer elements, the CRE and EGFRE and collectively referred to as the Mitogen Response Unit (MRU). We found that CRE is responsible for forskolin, cAMP and TPA whereas EGFRE is for EGF stimulation. We examined the minimal promoter and enhancer elements of the mouse lactoferrin gene that are required for EGF induced transcriptional activation. We found that the CRE and noncanonical TATA box (ATAAA) are the minimal promoter elements for basal activity of the CAT reporter construct, whereas, the EGFRE is needed for an additional activity induced by EGF in transiently transfected human endometrial carcinoma RL95-2 cells (RL95-2). The EGFRE, however, did not function in heterologous promoters (SV 40 and TK). Therefore, EGF-stimulated lactoferrin gene activity is promoter specific in RL95-2 cells. Mutation made at either elements or insertion of extra nucleotides between the two elements, severely affected EGF-stimulated activity. Nuclear protein prepared from RL95-2 cells protected the EGFRE, CRE and noncanonical TATA from DNAase I digestion in a footprinting analysis. Nuclear protein which interacted with the CRE were previously identified as API and CREB. In this study, we isolated a cDNA clone from an RL95-2 expression library that encodes the EGFRE binding protein. Partial sequence of the cDNA clone revealed 100% nucleotide identity with a GC-box binding protein, BTEB2. Protein-protein interaction among the transcription factors could fine-tune the mouse lactoferrin expression in various tissues.
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PMID:Mouse lactoferrin gene. Promoter-specific regulation by EGF and cDNA cloning of the EGF-response-element binding protein. 978 44

The proximal promoter and first intron of the fatty acid synthase (FAS) gene contains response sequences for insulin and glucose, but the 2- to 3-fold increase in FAS promoter activity attributable to these sequences falls short of the 20- to 30-fold induction in hepatic FAS gene transcription observed in fasted-refed rats. Using DNase I hypersensitivity site (HSS) mapping, two new liver specific sites were localized to the regions of: -8600 to -8500 (HSS 1) and -7300 to -7000 (HSS 2). DNase sensitivity of the -7300 to -7000 region was increased when fasted rats were refed glucose. When rat hepatocytes were transfected with a CAT construct that linked the region of -9700 and -4606 with the insulin response region located between -265 to +65, FAS promoter activity was induced 15-fold. This increase required the presence of both insulin and glucocorticoids. Deleting HSS 2 abolished the 15-fold induction in FAS promoter activity, but removing HSS 1 was without effect. Apparently the in vivo regulation of hepatic FAS gene transcription requires response elements located in the region of -7300 to -7000 and -265 to +65.
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PMID:Identification of a novel enhancer sequence in the distal promoter of the rat fatty acid synthase gene. 1042 97

We investigated gene transfer in finfish and shellfish via electroporated sperm. The mobility of sperm, the fertilization rate, the hatching rate, gene transfer rate, and abnormality rate of derived embryos were primarily dependent on the voltage level and concentration of DNA during electroporation. Optimal conditions for sperm of each species of aquatic animals can be reached. Genome of the electroporated sperm was analyzed by PCR, and it was shown that an expected-sized product was amplified, corresponding to that of the transgene's amplification. Southern blotting also showed that a positive band located at the same position as the DNA fragment used for the transfer was found in the electroporated sperm after DNase treatment. When the genome isolated from embryos, larvae, juvenile, and adult individuals, all derived from sperm electroporated with foreign DNA molecules, was analyzed by PCR, the existence of foreign DNA was detected in some samples. The integration of the transferred DNA into the genome of transgenic samples was also shown by Southern blot analysis. There was a mosaic distribution of exogenous DNA in a wide variety of tissues analyzed. In addition to CAT activity being positive for the experimental larvae, the transferred GH gene was functional in transgenic finfish and shellfish and resulted in fast-growing transgenic varieties. The overall evidence strongly suggests that the use of electroporated sperm is the simplest yet most efficient approach to perform mass gene transfer in aquacultural animals, including marine mollusks.
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PMID:Electroporated sperm mediation of a gene transfer system for finfish and shellfish. 1082 85

Phospholipases A(2) (PLA(2)s) catalyzing the hydrolysis of phospholipids form a family of proteins with diverse physiological and pharmacological properties. While there have been several reports on the cloning of PLA(2) cDNAs, very few studies have been carried out on the PLA(2) genes and, most importantly, no information has been available on the gene structure and function of group I venom PLA(2). This study, on the PLA(2) gene from a spitting cobra, besides being the very first report on any venom group I PLA(2) gene, constitutes the missing link in the biology and evolution of phospholipases. The 4-kb gene consists of four exons and three introns and resembles the human pancreatic PLA(2) gene. However, the size of intron 3 in particular is much smaller than that in the pancreatic gene. Interestingly, the information for the toxic and most of the pharmacological properties of the venom PLA(2) can be attributed to the end of exon 3 and the whole of exon 4 of the gene. This functional delineation fits in well with the theory of adaptive evolution exhibited by the venom PLA(2)s. We also show that the mammalian pancreatic and elapid PLA(2)s have similar paths of evolution (probably following gene duplication) from a common ancestral gene. Venom group II phospholipases, although evolved from the same ancestor, diverged early in evolution from the group I PLA(2) genes. Intriguingly, CAT reporter gene assays and DNase 1 footprinting studies on the promoter and its deletion constructs using CHO and HepG2 cell lines identified the possible involvement of cis elements such as Sp1, AP2, gamma-IRE, and (TG)(12) repeats in the expression of the gene in a tissue-specific manner.
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PMID:Structure and phylogeny of the venom group I phospholipase A(2) gene. 1088 14

Human connexin 26 gene (Cx26) has been considered a candidate suppressor gene in mammary epithelial cells. To explain regulatory mechanism of the Cx26, a DNase-1 hypersensitive 1.6 kb fragment upstream of the 5'-terminus of the gene was sequenced and assayed with CAT reporter system. The results showed that the 1.6 kb sequence had powerful promoter activity. The fragment contains two GT boxes (centering at -6158 and -6213 bp), and a TATA-less TTAAAA box (-6237/-6232 bp) which is the new promoter region of human Cx26.
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PMID:New Regulatory Sequence of Human Connexin 26 Gene. 1213 85

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The self-aggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the self-aggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (+/-) of 1/1; ACS/DNA complexes were formed at a lower charge ratio (+/-) of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C(2)C(12) cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.
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PMID:N-alkylated chitosan as a potential nonviral vector for gene transfection. 1286 31


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