Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Site-specific mutagenesis of the highly conserved milk box (-140 to -110) region suggested that beta-casein expression is regulated by a hormone-mediated relief of repression (M. Schmitt-Ney, W. Doppler, R. K. Ball, and B. Groner, Mol. Cell. Biol. 11:3745-3755, 1991). However, when this sequence was placed upstream of a heterologous thymidine kinase promoter, it activated reporter gene expression. This apparent paradox was resolved when the trans-acting factor YY1, capable of acting as both a positive and negative regulator, was shown to interact with the milk box region, using bacterially expressed YY1 and specific oligonucleotide and antibody competition experiments. Second, it was demonstrated that extracts prepared from several cell types contained a protein(s) interacting with the mammary gland-specific factor (MGF) binding site, previously shown to be required for beta-casein promoter activity (Schmitt-Ney et al., Mol. Cell. Biol. 11:3745-3755, 1991). Sequence analysis of this site revealed similarity to the gamma interferon-activated sequence, suggesting that MGF may be related to the stat91 signaling protein. Finally, using an oligonucleotide encompassing both the YY1 and MGF sites, we detected a slow-mobility complex only in extracts from mammary glands at late pregnancy and lactation (lactation-associated complex [LAC]). Site-specific mutation of the YY1 binding site led to an enhancement in LAC DNA binding activity, while mutation of the MGF site decreased detectable LAC. These results support a model in which lactogenic stimuli lead to a decrease in YY1 binding, and subsequent increased formation of LAC at a nearby binding site, to stimulate beta-casein transcription.
 ...
PMID:YY1 represses beta-casein gene expression by preventing the formation of a lactation-associated complex. 811 9

Prolactin (PRL) induces transcriptional activation of not only growth-related genes such as interferon regulatory factor-1 (IRF-1) but also differentiation-specific genes such as beta-casein through a signaling cascade consisting of Janus kinases and Stat (signal transducer and activator of transcription) factors. To understand better the role of Stats in PRL signaling, we cloned rat Stat5b from a PRL-responsive T cell line Nb2. A Stat5b-specific peptide antibody was generated. In PRL receptor reconstituted COS cells cotransfected with Stat5b or Stat5a, both Stat5 proteins become tyrosine phosphorylated and bind to the IRF-1 GAS (interferon-gamma activation sequence) element in a PRL-inducible manner. Unexpectedly, both Stat5b and Stat5a inhibit PRL induction of the IRF-1 promoter, but they mediate PRL stimulation of the beta-casein promoter. Stat5-mediated inhibition was observed only at the native IRF-1 promoter and not at the isolated IRF-1 GAS element linked to a heterologous thymidine kinase promoter. Mutational analyses showed that the DNA binding activity of Stat5b is not required, but the carboxyl-terminal transactivation domain is essential for Stat5b to inhibit PRL induction of the IRF-1 promoter. These results suggest that Stat5b mediates inhibition via protein-protein interactions. In contrast, both DNA binding and transactivation domains of Stat5b are required to mediate PRL induction of the beta-casein promoter. Furthermore, a carboxyl-terminal truncated dominant negative Stat5b can reverse Stat5b inhibition at the IRF-1 promoter. These studies suggest that Stat proteins can act as not only positive but also negative regulators of gene transcription. Further, Stat5 can modulate gene expression without binding to DNA but via protein-protein interactions.
 ...
PMID:Transcriptional inhibition by Stat5. Differential activities at growth-related versus differentiation-specific promoters. 934 Nov 15

GH and PRL stimulate insulin production in pancreatic beta-cells through induction of insulin gene transcription. The transcriptional effects of GH are mediated through the binding of signal transducer and activator of transcription-5 (STAT5) to a consensus recognition sequence (TTCnnnGAA) in the rat insulin-1 promoter. In this study we demonstrate that PRL also induces the binding of STAT5 proteins to the rat insulin-1 STAT5 motif. However, the magnitude of binding of STAT5 nuclear proteins, as assessed by electrophoretic mobility shift assays, was only 1/30th that of the binding of the same STAT5 proteins to the beta-casein STAT5 site. The differences in the affinities of the rat insulin-1 and beta-casein STAT5 motifs are explained in part by differences in promoter sequences flanking the STAT5 sites. To assess the importance of the STAT motif in PRL induction of insulin gene transcription, we deleted the STAT5 consensus sequence in the rat insulin 1 promoter, cloned the truncated promoter upstream of the luciferase reporter gene, and transfected the construct into rat insulinoma (INS-1) cells. The transcriptional activity of this construct was compared with that of the wild-type promoter. Although deletion of the STAT5 site in the promoter reduced the basal luciferase activity, the response to PRL was unaffected. PRL also induced transcription of constructs containing the wild-type human insulin promoter or the rat insulin-2 promoter, which contain no classic STAT5 sequences. The transcriptional effect of PRL was manifest even when cells were incubated in glucose-free medium, indicating that the action of the hormone is not mediated solely through changes in glucose uptake or glucose metabolism. To identify PRL-responsive regions of the rat and human insulin promoters, we constructed a series of promoter truncations and assessed their responsiveness to PRL. A PRL-responsive region of the rat insulin-1 promoter was localized between nucleotides -165 and -109. A PRL-responsive region of the human insulin promoter was localized between nucleotides -346 and -250. Additional regions of the human and rat insulin-1 promoters were required for PRL induction of a heterologous, minimal thymidine kinase promoter, suggesting that there are multiple PRL-responsive elements in the insulin genes. These observations suggest a glucose- and STAT5-independent pathway by which PRL may induce insulin gene transcription.
 ...
PMID:Prolactin induction of insulin gene transcription: roles of glucose and signal transducer and activator of transcription 5. 1141 99

The production of recombinant protein is one of the major successes of biotechnology, animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animal mammary gland bioreactor are being used for this purpose. Gene targeting is a more powerful method to produce mammary gland bioreactor, and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe efficient and reproducible gene targeting in goat fetal fibroblasts to place the human tissue plasminogen activator mutant (ht-PAm) cDNA at the beta-casein locus, and would produce the transgenic goat by nuclear transfer. To construct the gene targeting vector pGBC4tPA, the milk goat beta-casein genomic DNA sequence for homologous arms had been cloned firstly. The left arm was 6.3 kb fragment including goat beta-casein gene 5' flanking sequence, and the right arm was 2.4 kb fragement including beta-casein gene from exon 8 to exon 9. The ht-PAm cDNA was subcloned in the goat beta-casein gene exon 2, and the endogenous start condon was replaced by that of ht-PAm. The bacterial neomycin (neo) gene as positive selection marker gene, was placed in the beta-casein gene intron 7, the thymidine kinase (tk) as the negative selection marker gene, was just outside the right arm. The validity of the positive-negative selection vector (PNS), was tested, and targeting homologous recombination (HR) were elevated to 5-fold with the negative selection marker using the drug GANC. The DNA fragment in which two LoxP sequence was delected effectively using Cre recombinase in vitro. Goat fetal fibroblasts were thawed and cultured to subconfluence before transfection, about 10(7) fibroblasts were electoporated at 240V, 600 microF in 0.8 mL PBS buffer containing linear pGBC4tPA. transfected cells were cultured in collagen-coated 96-wellplate for 24h without selection, then added the drug G418 (600 microg/mL) and GANC (2 micromol/L). After 12 days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate. 244 clones were selected, and only 90 clones could grow and be tested by PCR screening for targeting. The primary result demonstrated that 31 targeting cell clones with homologous recombination events were obtained, and 2 cell clones was verified by DNA sequence analysis on the homologous recombination region.
 ...
PMID:[The ht-PAm cDNA knock-in the goat beta-casein gene locus]. 1597 6

Gene targeting is a more powerful method to produce mammary gland bioreactor and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe an efficient and reproducible gene targeting in goat mammary epithelium cell to place the GFP and neo at the beta-casein locus. The transgenic goat would be produced by nuclear transfer. To construct the gene targeting vector pGBC-GFP-neo, the milk goat beta-casein genomic DNA sequence for homologous arms was cloned first. The left arm was 2.1 kb fragment including goat beta-casein gene exon1 and part of exon 2, and the right arm was 5.1 kb fragment including beta-casein gene from exon 7 to 3'-flanking sequence. The bacterial neomycin (neo) gene as positive selection marker gene, with the promoter-trap GFP, was placed between two loxPs. The thymidine kinase (tk) as negative selection marker gene was just outside the right or left arms. Goat mammary epithelium cells were cultured to sub-confluence about 90% and transfected with linear pGBC-GFP-neo using Lipefectamin-2000. These transfected cells were cultured in collagen-coated 96-wellplate for 24 h without selection, then added the drug G418(600 microg/mL) and GANC(2 micromol/L). After nine days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate; 51 clones were selected; 17 clones were tested by GFP expression using promoter-trap strategy; only four clones grow well. After PCR confirmation the four targeting cell clones homologous recombination were used as the donor cell for nuclear transfer. 59.5% cloned embryos could develop up. Some could develop to morula or blastocyst in vitro.
 ...
PMID:[High-efficient gene targeting of goat mammary epithelium cell by the multi-selection mechanism]. 1601 Oct 27