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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gene constructs consisting of human growth hormone (hGH) gene driven by promoter/regulatory sequence of mouse metallothionein (mMT), viral thymidine kinase (vTK), rat cholecystokinin (rCCK), or chicken beta-actin (cBA) gene were injected into the cytoplasm of fertilized medaka eggs via the micropyle. More than 49% of the injected embryos survived at hatching. Up to 26% of the survivors showed integration of the introduced gene construct, as determined by polymerase chain reaction analysis and subsequent confirmation by Southern blot hybridization of the genomic DNA. A significant fraction of F1 progeny, derived from crosses between transgenic founders and the nontransgenic individuals, inherited the transgene. Expression of hGH gene was also observed in some of the P1 founders and F1 transgenic progeny carrying mMT-hCG or cBA-hGH gene. Furthermore, the growth performance of the P1 mMT-hGH and cBA-hGH transgenic founders and F1 cBA-hGH F1 transgenic progeny was significantly greater than their full sibling, nontransgenic individuals. In addition to the microinjection experiment, a gene construct containing the long-terminal repeat (LTR) sequence of avian Rous sarcoma virus (RSV) and rainbow trout (rt) GH2 cDNA was introduced into embryos of medaka by electroporation using an exponential decay electroporator. Approximately 70% of the electroporated embryos survived at hatching, and 20% of the survived individuals integrated RSVLTR-rtGH2 cDNA into their genomes. These two techniques will greatly enhance the ability to study regulation of gene expression in transgenic animals during differentiation and development.
Mol Mar Biol Biotechnol
PMID:Integration, expression and germ-line transmission of foreign growth hormone genes in medaka (Oryzias latipes). 128 9

We describe conditions under which exogenous DNA templates can be introduced for transient expression into primary murine T lymphocytes. T cells at various stages of development, including concanavalin A-activated splenic T cells, immature pre-T cells, and even small cortical thymocytes, could be successfully transfected. A variety of model DNA constructs were compared in which different viral promoter regions were used to drive expression of the chloramphenicol acetyltransferase (CAT) reporter gene. All showed enhanced expression in cells that had been acutely stimulated with the Ca2+ ionophore A23187 and phorbol ester as chemical proxies for T-cell receptor-mediated signals. In addition, splenocytes but not thymocytes required prior treatment with a mitogen and interleukin-2 in order to express these constructs, implying that even postmitotic thymocytes may be held in a quasiactivated state. A most striking result was the finding that the viral regulatory sequences in the Rous sarcoma virus long terminal repeat and the simian virus 40 early region were subject to sharply differential regulation, with a rank order that changed depending on the developmental stage of the T cells. The most immature thymic blasts and several lymphoma cell lines expressed the pRSV-Cat and pSV2-Cat constructs similarly, but cortical thymocytes exhibited a strong preference for pSV2-Cat. Splenic concanavalin A-stimulated blasts, on the other hand, slightly preferred pRSV-Cat, a tendency which became exaggerated in factor-dependent T-cell lines. The ratio of pRSV-Cat to pSV2-Cat expression varied according to cell type by as much as 500-fold. These results argue against a trivial linkage of promoter preference to cell cycle status but instead provide evidence that activation of T cells at distinct stages of differentiation results in the expression of different ensembles of nuclear regulatory proteins. In contrast to the simian virus 40 and Rous sarcoma virus promoter regions, the long terminal repeats of the retroviruses mink cell focus-forming virus and Akv were expressed well in all primary T-lineage cells. Thus, they represent excellent model promoters for engineering developmental stage-independent expression of exogenous genes in murine T cells.
Mol Cell Biol 1992 Apr
PMID:In vitro transfection of fresh thymocytes and T cells shows subset-specific expression of viral promoters. 131 65

Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biol 1992 May
PMID:Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. 131 54

The EFII cis element is a 38-bp sequence at the 5' end of the Rous sarcoma virus long terminal repeat, extending from nucleotides -229 to -192 (with respect to the viral transcription start site), which is recognized by sequence-specific DNA-binding proteins in avian fibroblast nuclear extracts (L. Sealy and R. Chalkley, Mol. Cell. Biol. 7:787-798, 1987). We demonstrate that multiple copies of the EFII cis element strongly activate transcription of a reporter gene in vivo. We correlate the region of the EFII cis element which activates transcription in vivo with the in vitro binding site for three nuclear factors, EFIIa, EFIIb, and EFIIc. The sequence motif recognized by EFIIa, -b, and -c is also found in consensus binding sites for members of a rapidly growing family of transcription factors related to the CCAAT/enhancer-binding protein (C/EBP). EFIIa, -b, and -c are present in fibroblast and epithelial cell lines from various species but are much less abundant in differentiated rat liver and kidney cells. The EFIIa binding activity is particularly abundant in an avian B-cell lymphoma line. As judged from molecular weight analysis, cell type distribution, and sequence recognition properties, the EFII factors under study appear to differ from most of the previously described C/EBP-related factors and thus may expand the diversity of the C/EBP family.
 ...
PMID:Characterization of nuclear proteins that bind the EFII enhancer sequence in the Rous sarcoma virus long terminal repeat. 132 70

We have previously reported on the presence of a CArG motif at -100 in the Rous sarcoma virus long terminal repeat which binds an avian nuclear protein termed enhancer factor III (EFIII) (A. Boulden and L. Sealy, Virology 174:204-216, 1990). By all analyses, EFIII protein appears to be the avian homolog of the serum response factor (SRF). In this study, we identify a second CArG motif (EFIIIB) in the Rous sarcoma virus long terminal repeat enhancer at -162 and show only slightly lower binding affinity of the EFIII/SRF protein for this element in comparison with c-fos serum response element (SRE) and EFIII DNAs. Although all three elements bind the SRF with similar affinities, serum induction mediated by the c-fos SRE greatly exceeds that effected by the EFIII or EFIIIB sequence. We postulated that this difference in serum inducibility might result from binding of factors other than the SRF which occurs on the c-fos SRE but not on EFIII and EFIIIB sequences. Upon closer inspection of nuclear proteins which bind the c-fos SRE in chicken embryo fibroblast and NIH 3T3 nuclear extracts, we discovered another binding factor, SRE-binding protein (SRE BP), which fails to recognize EFIII DNA with high affinity. Competition analyses, methylation interference, and site-directed mutagenesis have determined that the SRE BP binding element overlaps and lies immediately 3' to the CArG box of the c-fos SRE. Mutation of the c-fos SRE so that it no longer binds SRE BP reduces serum inducibility to 33% of the wild-type level. Conversely, mutation of the EFIII sequence so that it binds SRE BP with high affinity results in a 400% increase in serum induction, with maximal stimulation equaling that of the c-fos SRE. We conclude that binding of both SRE BP and SRF is required for maximal serum induction. The SRE BP binding site coincides with the recently reported binding site for rNF-IL6 on the c-fos SRE. Nonetheless, we show that SRE BP is distinct from rNF-IL6, and identification of this novel factor is being pursued.
Mol Cell Biol 1992 Oct
PMID:Maximal serum stimulation of the c-fos serum response element requires both the serum response factor and a novel binding factor, SRE-binding protein. 132 62

The Schmidt-Ruppin or the B77 strain of Rous sarcoma virus (RSV) was inoculated into limb buds of 4.5-days-old avian embryos. No sarcoma but blister formation was observed in those RSV-inoculated embryos. Protein kinase activity of pp60v-src in RSV-inoculated embryos, even in the site of virus inoculation, was the same as that in mock-infected embryos. This indicated that the expression of the v-src gene did not attain superiority over that of the c-src gene in RSV-inoculated embryos. The v-src gene was detected in every DNA from tissues of RSV-inoculated embryos but not in DNAs from tissues of RSV-inoculated chicken except for the DNA from Rous sarcoma. Those results confirmed that the lack of sarcoma induction in early avian embryos by RSV was due to the lack of the expression of the v-src gene which was present in the target cells.
Cell Mol Biol (Noisy-le-grand)
PMID:Rous sarcoma virus does not induce sarcoma in early chick embryos by lack of the v-src gene expression. 133 25

We report the fish use of an exponential decay electroporation system to introduce foreign DNA into fertilized zebrafish embryos. The plasmid RSVCAT (Rous sarcoma viral promoter (RSV) upstream from the chloramphenicol acetyltransferase gene (CAT)) was linearized and introduced into fertile zebrafish embryos by electroporation no later than the four-cell stage. Conditions for the procedure were empirically derived, and 68% of the treated animals survived through hatching to at least 6 days after fertilization and well beyond. Dot-blot analysis on DNA extracted from individual hatching fry demonstrated that 65% of the animals tested carried the foreign construct. Enzyme assays on the soluble proteins of treated animals were positive for chloramphenicol acetyltransferase activity. These data demonstrate that the foreign construct was being transiently expressed in the developing tissues of the embryo. The simplicity of this technique will greatly enhance the ability to analyze gene promoter regulation in vivo in transgenic zebrafish. The ability of the electroporated DNA to integrate into the host genome and to generate stable lines of transgenic fish is discussed.
Mol Mar Biol Biotechnol
PMID:Transient expression of RSVCAT in transgenic zebrafish made by electroporation. 133 27

Recombinant plasmids containing the Rous sarcoma virus long-terminal repeat (RSVLTR) promoter linked to either rainbow trout (Oncorhyncus mykiss) growth hormone 1 (rtGH1) or growth hormone 2 (rtGH2) cDNA were linearized and introduced into the fertilized eggs of zebrafish (Brachydanio rerio), channel catfish (Ictalurus punctatus), and common carp (Cyprinus carpio) by both electroporation and microinjection. The latter two species had these rainbow trout constructs (RSVLTR-rtGH1cDNA or RSVLTR-rtGH2) electroporated into both gametes (i.e., sperm and unfertilized eggs) prior to fertilization, into eggs shortly after fertilization, and at the first cell division stage. Survival was determined just after hatching and again between 3 and 5 months after hatching. Polymerase chain reactions and Southern blot analyses were used to detect those individuals carrying the introduced foreign genes 3 to 5 months after hatching, respectively. Individuals analyzed by both methods yielded identical results in a double-blind study. The electroporation results were compared with groups that were microinjected. Although survival was similar, electroporation tended to produce a greater number of transgenic individuals than the microinjection procedure, and many more eggs could be treated per unit time by electroporation than microinjection. Survival was better for common carp when electroporation was performed shortly after fertilization, whereas channel catfish fared better at the first cell division stage. Electroporation prior to and shortly after fertilization, and at the first cell stage appeared to generate a large fraction of transgenic fish. We cautiously conclude that electroporation is an efficient method for introducing foreign DNA into fish gametes and embryos and may be an ideal method for treating large numbers of gametes in a modest period.
Mol Mar Biol Biotechnol
PMID:Electroporation: a method for transferring genes into the gametes of zebrafish (Brachydanio rerio), channel catfish (Ictalurus punctatus), and common carp (Cyprinus carpio). 133 28

The angiotensinogen gene encodes the precursor protein for the potent vasoconstrictor angiotensin II. Although the gene is expressed in several tissues, the liver is the major source of circulating protein. In previous in-vivo studies we have found that a mini-gene containing 750 bp of 5'-flanking sequence is transcribed in a manner which largely parallels the expression of the endogenous gene. In this report, we characterized conserved elements in the promoter region, in order to determine their role in the transcription of the angiotensinogen gene. Constructs fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into hepatocarcinoma Hep G2 cells as well as into nonhepatic cell lines. We found that 5'-deletion mutant constructs, containing sequences from +25 to -90 bp and -321 to -750 bp, were each able to activate transcription. These constructs contain the TATA box and core promoter sequences, including an Sp1-binding site, and two glucocorticoid responsive elements respectively. In the non-hepatic cell lines, HeLa and Jeg-3, we found that the constructs were transcribed at a much lower rate when compared with the expression of a plasmid containing the Rous sarcoma virus long terminal repeat fused to the CAT gene. Constructs which included sequence 5' to -244 were oestrogen inducible. An element which is conserved between rodent and human angiotensinogen promoters is contained within a sequence which is oestrogen responsive, while another binds the liver-enriched transcriptional activator hepatocyte nuclear factor 1. However, the role of this transactivator in the transcription of angiotensinogen remains uncertain.
J Mol Endocrinol 1992 Aug
PMID:The function of conserved elements in the promoter of the mouse angiotensinogen gene. 151 23

The CEF-4/9E3 gene is expressed constitutively in Rous sarcoma virus (RSV)-transformed cells. This expression is largely determined by an increase in transcription of the gene. In this report, we characterize the regulatory elements responsible for the transformation-dependent activation of CEF-4/9E3. Three sequences corresponding to AP-1, PRD II/kappa B, and TAACGCAATT are involved in the process and therefore define the src-responsive unit (SRU) of the CEF-4 promoter. In constructs containing a deletion of the SRU, multiple copies of AP-1 or PRD II/kappa B, but not TAACGCAATT, led to activation of the promoter. Thus, factors interacting with these elements are constitutively activated in RSV-transformed chicken embryo fibroblasts. In agreement with the results of transient expression assays, protein binding to AP-1, PRD II/kappa B, and TAACGCAATT were more abundant in the nuclei of transformed cells. The expression of the CEF-4 promoter was investigated in cells infected by a temperature-sensitive mutant of RSV. No significant increase in CEF-4 promoter activity was detected early after activation of pp60v-src. In contrast, a substantial activation of the CEF-4 promoter was detected late after a temperature shift. Factors interacting with the TAACGCAATT, PRD II/kappa B, and AP-1 elements accumulated gradually over a period of several hours. Therefore, transcriptional activation plays an important role in the late, constitutive expression of the CEF-4 gene in stably transformed cells.
Mol Cell Biol 1992 Apr
PMID:Transcriptional activation of the CEF-4/9E3 cytokine gene by pp60v-src. 154 6


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