Mapping of the murine nuclear factor I/X gene (Nfix) to mouse chromosome 8 C1-2 by FISH.
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Animals↗
Biomedical subjects
E L Winnacker
Publications and source records attributed to E L Winnacker.
Transcriptional activation by CTF proteins is mediated by a bipartite low-proline domain.
Members of the CCAAT-binding transcription factor (CTF) family of proteins stimulate the initiation of adenovirus DNA replication and act as transcriptional activators. To investigate the mechanisms underlying CTF-mediated transactivation patterns, we expressed several natural CTF variants in Saccharomyces cerevisiae and determined their transactivating activities in enzymatic assays. CTF7, which lacks the entire proline-rich region previously thought to mediate transcriptional activation by CTF proteins, enhances transcription to a greater degree than full-length CTF1, which contains the putative activation domain. CTF2, which contains a partially deleted proline-rich activation region, does not stimulate transcription at all. These findings indicate that the proline-rich region of CTF proteins is not essential for transcriptional activation in yeast. Our studies also suggest a bipartite two-domain structure of CTF-type transcriptional activation domains.
Alternative Splicing↗
A putative homologue of the human autoantigen Ku from Saccharomyces cerevisiae.
We have identified and purified a new DNA binding protein, designated HDF (high affinity DNA-binding factor) from Saccharomyces cerevisiae. HDF binds in a sequence-independent manner to the ends of double-stranded DNA. The protein appears as a stable heterodimer of two polypeptides with molecular masses of 70 and 85 kDa. We have cloned and sequenced the 70-kDa subunit of the HDF protein. The amino acid sequence shows a weak but significant homology with the p70 subunit of the human Ku autoantigen, a protein that also binds to the ends of double-stranded DNA. Hdf- strains generated by one-step gene disruption show a temperature-sensitive phenotype for growth at 37 degrees C. Cells arrest growth at 37 degrees C and after several hours appear as enlarged single-budded cells with abnormally high DNA content indicating a defect in the regulation of DNA replication coupled with or causing a cell cycle arrest in G2 or mitosis.
Amino Acid Sequence↗
Localisation of the human nuclear factor I/X (NFI/X) gene to chromosome 19p13 and detection of five other related loci at 1p21-22, 1q42-43, 5q15, 11p13 and 20q13 by FISH.
Nuclear factor I (NFI) is a member of a family of dimeric DNA-binding proteins that are involved both in the initiation of adenovirus DNA replication and in the stimulation of transcriptional activation. We have used fluorescence in situ hybridisation (FISH) to map one of four known genes encoding an NFI protein, the human NFI/X gene, to chromosome 19p1.3. Secondary sites of hybridisation observed at 5p1.5, 1q4.2-4.4, 1p2.1-2.2, and 20p1.3 most likely are attributable to partial sequence homologies with related NFI genes.
CCAAT-Enhancer-Binding Proteins↗
Transgenic pigs carrying cDNA copies encoding the murine Mx1 protein which confers resistance to influenza virus infection.
An important aspect of gene transfer into farm animals is the improvement of disease resistance. The mouse Mx1 protein is known to be sufficient to confer resistance to influenza viruses. Gene constructs containing the mouse Mx1 cDNA controlled by the human metallothionein IIA promoter (hMTIIA::Mx), the SV40 early enhancer/promoter region (SV40::Mx) and the mouse Mx1 promoter (mMx::Mx) were transferred into pigs. The results of the gene transfer experiments with the hMTIIA::Mx and the SV40::Mx constructs indicate that the permanent high-level synthesis of Mx1 might be deleterious to the organism: the gene transfer efficiency was surprisingly low, and all transgenic piglets born had rearrangements in their transgene copies that abolished protein synthesis. The use of the interferon (IFN)- and virus-inducible mMx::Mx construct resulted in normal gene transfer efficiency. Two transgenic pig lines could be established which expressed IFN-inducible mouse Mx1 mRNA. Extensive protein analysis did not detect mouse Mx1 in IFN-treated transgenic animals.
Animals↗
Nuclear factor I (NF I) binds to an NF I-type site but not to the CCAAT site in the human alpha-globin gene promoter.
Methylation interference and missing contact analyses demonstrate that nuclear factor I (NF I) recognizes an NF I-like site (5'-GGG(N)6GCCAG-3') within the alpha-globin promoter rather than the adjacent CCAAT box. Consistent with this, mutations within the CCAAT box do not alter significantly the affinity and specificity of the interaction whereas elimination of the 5'-GGG-3' half-site of the recognition sequence reduces the DNA binding strength of NF I by 2 orders of magnitude down to the range of unspecific interaction. On the other hand, the mutated alpha-globin promoter sequence that is no longer bound by NF I, although it retains an intact CCAAT box, interacts specifically with a protein component from nuclear extracts of HeLa cells. From these results we conclude that NF I is not the factor that interacts with the CCAAT box and that the second half of the canonical 5'-TGG(N)6GCCAA-3' NF I binding site cannot be regarded as identical with the CCAAT promoter element, as suggested previously.
Autoradiography↗
Molecular cloning of porcine Mx cDNAs: new members of a family of interferon-inducible proteins with homology to GTP-binding proteins.
Porcine cells treated with interferon (IFN) or double-stranded RNA synthesize two proteins that exhibit high homology of the amino acid sequence to mouse Mx1 protein involved in selective resistance to influenza virus. A full-length cDNA clone (poMx1) encoding the porcine Mx1 protein was isolated and sequenced. It contained an open reading frame of 663 amino acids. The predicted molecular weight of 75.6 kD is in good agreement with the apparent molecular mass of the two immunoprecipitable proteins of 76 kD and 73 kD determined by SDS polyacrylamide gel electrophoresis. A second cDNA (poMx2) was characterized which was incomplete in the 5' region. A comparison of all known Mx proteins revealed an average homology of 67.5%. The porcine Mx1 polypeptide is most closely related to human MxA (p78), murine Mx2, rat Mx2, and rat Mx3 proteins. The amino-terminal halves of all Mx proteins are highly conserved and possess three consensus elements in proper spacing, characteristic of GTP-binding domains. The Mx family shows in their amino termini striking homology to previously characterized Mx-related proteins playing roles in the intracellular vectorial transport of proteins--the products of the yeast Vps1 locus and the dynamins.
Amino Acid Sequence↗
Two cellular single-strand-specific DNA-binding proteins interact with two regions of the bovine papillomavirus type 1 genome, including the origin of DNA replication.
We have identified and purified to near homogeneity two specific single-stranded DNA-binding factors (SPSF I and II) with molecular masses of 42 and 39 kDa, respectively, from calf thymus. Gel retention analysis and competition experiments demonstrate that the ubiquitous proteins SPSF I and II specifically interact with single-stranded DNA derived from the minimal in vitro origin of replication of bovine papillomavirus type 1 and a region of the viral genome proposed to be involved in plasmid maintenance. Bovine papillomavirus type 1 proteins do not interfere with DNA binding of SPSF I and II. The exact location of the binding domains of SPSF I and II on the DNA has been determined by methylation interference and T4 DNA polymerase footprinting. A potential cellular binding site for SPSF I and II is the major promoter (P2) of the human c-myc gene.
3T3 Cells↗
[Genetic techniques and medicine].
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Abortion, Eugenic↗
Reactivation of metal-requiring apoenzymes by phytochelatin-metal complexes.
The enzymatically inactive, metal-requiring apoforms of diamino oxidase and of carbonic anhydrase were reactivated by copper-and zinc-phytochelatin complexes, respectively. The level and the rate of reactivation effected by metal complexes consisting of poly(gamma-glutamylcysteinyl)glycine as well as by the respective free metal ion were compared. An efficient transfer of zinc and copper from phytochelatin-complexes to apoenzymes was observed in vitro.
Amine Oxidase (Copper-Containing)↗
The NF-kappa B transcription factor induces DNA bending which is modulated by its 65-kD subunit.
This study describes a novel functional property of the eukaryotic transcription factor NF-kappa B in altering the DNA structure. We observed that the binding of purified NF-kappa B to DNA was facilitated by spermine and cations which are known to promote DNA bending, and by a nick in one position at the 3'-end of the binding site. Furthermore, the position of the NF-kappa B binding sequence 5'-GGGACTTTCC-3' (kappa B motif) within circularly permutated DNA fragments had a profound influence on the mobility of NF-kappa B-DNA complexes in a gel retardation assay while the mobility of unbound DNA fragments did not depend on the position of the kappa B motif. The mobility effect was slightly reduced at increased temperature. These observations suggested that binding of NF-kappa B to DNA induces bending. The estimated bending angle induced by the 50-kD DNA binding subunit of NF-kappa B (p50) was smaller than that induced by p50 which was associated with the 65-kD non-DNA binding subunit (p65). Moreover, the presence of p65 appeared to result in a shift of the bending center from the middle towards the 3'-end of the kappa B motif. This shows a role for the p65 subunit of NF-kappa B in modulating the extent and altering the position of protein-induced DNA bending.
Animals↗
Transfer-RNA interferes with the uniform cleavage pattern of DNA by hydroxyl radicals.
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Base Sequence↗
Klenow polymerase is preferable to T4 polymerase in end-labeling reactions.
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Adenoviruses, Human↗
Nuclear factor I acts as a transcription factor on the MMTV promoter but competes with steroid hormone receptors for DNA binding.
Several steroid hormones induce transcription of the mouse mammary tumor virus (MMTV) promoter, through an interaction of their respective receptors with the hormone responsive elements (HREs) in the long terminal repeat (LTR) region. The molecular mechanism underlying transcriptional activation is not known, but binding of nuclear factor I (NFI) to a site adjacent to the HRE appears to be required for efficient transcription of the MMTV promoter. In JEG-3 choriocarcinoma cells the MMTV promoter is transcribed inefficiently, even after transfection of the receptor cDNA and treatment with glucocorticoids or progestins. These cells contain low levels of NFI as cotransfection of NFI cDNA enhances MMTV transcription and this effect is inhibited by mutation of the NFI binding site. In DNA binding experiments with purified NFI from pig liver, the glucocorticoid and progesterone receptors do not co-operate but rather compete with NFI for binding to their respective sites on the LTR. Similar results are obtained with a functional recombinant NFI synthesized in vitro. Competition for DNA binding is probably due to steric hindrance as the DNase I footprints of the hormone receptors and NFI do overlap. These results suggest that, though NFI acts as a transcription factor on the MMTV promoter, transcriptional activation does not take place through a direct facilitation of DNA binding of NFI by steroid hormone receptors.
Animals↗
Transgenic offspring by transcaryotic implantation of transgenic ovaries into normal mice.
Female transgenic mice may be unable to reproduce successfully if the product encoded by the transgene results in pathological changes or affects the fertility of the mouse. To approach this problem, we have produced chimaeras by transferring the ovaries of transgenic mice into normal mice of the same strain. Such chimaeras will be an ideal tool for investigating the interactions between transgenic ovaries and normal mice or vice versa. Here we show that, using this method, we were able to get large numbers of transgenic offspring even from founder transgenic female mice that were themselves infertile as a result of the overexpression of growth hormone genes. Although none of the ovary recipients were given immunosuppressant treatment, 60% of the recipients had biologically active ovaries over a mean period of about 100 days.
Animals↗
Hydroxyl radical footprints reveal novel structural features around the NF I binding site in adenovirus DNA.
We have identified a number of as yet unknown structural abnormalities of the NF I-DNA binding site within the inverted terminal repetition of adenovirus DNA by probing it with a hydroxyl radical footprinting technique. NF I binding alters the accessibility of the deoxyribose moieties to hydroxyl radicals both at the 3' and at the 5' side of the recognition sequence 5'-TGG(N)6GCCAA-3'. A smooth bend at the 5' side of the binding sequence is already present in naked linear DNA and it is further enhanced by protein binding. This could be demonstrated not only by hydroxyl radical footprinting but also by studying the temperature dependent mobility during gel electrophoresis of DNA fragments carrying the NF I binding site at circularly permutated positions. We propose that the bent conformation at this site is responsible for facilitating protein/DNA interactions.
Adenoviruses, Human↗
Structural and functional organization of a porcine gene coding for nuclear factor I.
This paper describes the structure of a 70-kb porcine gene for nuclear factor I, including its promoter region, comprising a total of 11 exons. Different mRNAs that we have isolated as cDNAs from both porcine liver and human HeLa cells presumably are generated from this gene by differential splicing events. One cDNA species from porcine liver that lacks exon 9 carries coding information for a protein of 439 amino acids. The in vitro translated protein displays all the properties of an NFI-like protein with high affinity toward the sequence element TGG(N)6GCCAA, as shown by gel shift analysis, and no or little affinity toward CCAAT box containing sequences. Cotranslation experiments with full-length and truncated variants of the protein demonstrate that it binds as a dimer to its cognate DNA recognition sequence. Its DNA-binding domain which is retained in all cDNA clones was mapped by deletion analysis to the 250 N-terminal amino acids of the protein. No structural homologies are observed between this protein and other known DNA-binding proteins; instead, the protein contains a novel alpha-helical sequence motif consisting of several lysine residues spaced at intervals of seven amino acids which we have termed the "lysine helix". The C-terminal portion of the protein derived from full-length cDNAs encodes a short amino acid sequence which is identical with the heptapeptide repeat CT7 observed in the C-terminal domain of the largest subunits of yeast and mouse RNA polymerase II. This region is removed by differential splicing in some of the NFI/CTF cDNAs and thus may be of functional significance.
Amino Acid Sequence↗