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W Schaffner

Publications and source records attributed to W Schaffner.

At least 127 records · Page 7Linked to original sources
PubMed1995

The leucine zipper of c-Jun binds to ribosomal protein L18a: a role in Jun protein regulation?

Recently we developed a method called direct interaction rescue (DIRE) for selective cloning in filamentous phage. The rescue is effected by the interaction of two heterologous proteins, one fused to the N-terminus of gene 3 adhesion protein, the other fused to the C-terminus. When heterologous fusion proteins interact with each other, gene 3 protein activity in restored thereby rescuing phage infectivity. We have used the leucine zipper of c-Jun protein as a 'bait' to select for interacting proteins from a human cDNA library. Two interacting clones were isolated, one coding for ribosomal protein L18a, a component of the large ribosomal subunit, and the other for tropomyosin, a component of the cytoskeleton. L18a contains two zipper-like domains which probably interact with c-Jun. We consider it possible that L18a (and tropomyosin) are involved in the cellular regulation of Jun protein levels.

Amino Acid Sequence↗
PubMed1994

The CpG-specific methylase SssI has topoisomerase activity in the presence of Mg2+.

A prokaryotic CpG-specific methylase from Spiroplasma, SssI methylase, is now widely used to study the effect of CpG methylation in mammalian cells, and can processively modify cytosines in CpG dinucleotides in the absence of Mg2+. In the presence of Mg2+, we found (i) that the methylation reaction is distributive rather than processive as a result of the decreased affinity of SssI methylase for DNA, and (ii) that a type I-like topoisomerase activity is present in SssI methylase preparations. This topoisomerase activity was still present in SssI methylase further purified by either SDS-polyacrylamide or isoelectric focusing gel electrophoresis. We show that methylase and topoisomerase activities are not functionally interdependent, since conditions exist where only one or the other enzymatic activity is detectable. The catalytic domains of SssI methylase and prokaryotic topoisomerases show similarity at the amino acid level, further supporting the idea that the topoisomerase activity is a genuine activity of SssI methylase. Mycoplasmas, including Spiroplasma, have the smallest genomes of all living organisms; thus, this condensation of two enzymatic activities into the same protein may be a result of genome economy, and may also have functional implications for the mechanism of methylation.

Amino Acid Sequence↗
PubMed1994

Strong transcriptional activators isolated from viral DNA by the 'activator trap', a novel selection system in mammalian cells.

Transcription factors often contain activation domains that interact with the basic transcription machinery. We have developed a functional screening strategy in mammalian cells to selectively isolate activation domains from a library of random DNA inserts. For this, sonicated DNA fragments are cloned next to the DNA binding domain of GAL4 factor in a plasmid that also contains the SV40 origin of replication. Pools of fusion protein clones are transfected into CV-1-5GT monkey cells containing an SV40 T antigen gene under the control of a promoter with GAL4 binding sites. Plasmids that express functional transactivating fusion proteins activate the T antigen gene, thus promoting selective amplification of the plasmid in the mammalian host cell line. Using this method, we were able to select strong enhancer-type activation domains from the immediate early regions of two herpesviruses, namely pseudorabies virus and bovine herpesvirus 1. In both cases, the activation domains selected were homologues of the ICP4 regulatory protein of herpes simplex virus. The activation domain from pseudorabies virus is four times stronger than the activation domain of herpes simplex virus protein VP16 (Vmw65), making it the strongest activation domain characterized so far. This activator trap method should be useful for precisely localizing activation domain(s) in known factors, or to identify mammalian transcriptional adaptors that do not bind DNA and which may escape conventional detection methods.

Amino Acid Sequence↗
PubMed1994

Cloning, chromosomal mapping and characterization of the human metal-regulatory transcription factor MTF-1.

Metallothioneins (MTs) are small cysteine-rich proteins that bind heavy metal ions such as zinc, cadmium and copper with high affinity, and have been functionally implicated in heavy metal detoxification and radical scavenging. Transcription of metallothioneins genes is induced by exposure of cells to heavy metals. This induction is mediated by metal-responsive promoter elements (MREs). We have previously cloned the cDNA of an MRE-binding transcription factor (MTF-1) from the mouse. Here we present the human cDNA equivalent of this metal-regulatory factor. Human MTF-1 is a protein of 753 amino acids with 93% amino acid sequence identity to mouse MTF-1 and has an extension of 78 amino acids at the C-terminus without counterpart in the mouse. The factors of both species have the same overall structure including six zinc fingers in the DNA binding domain. We have physically mapped the human MTF-1 gene to human chromosome 1 where it localizes to the short arm in the region 1p32-34, most likely 1p33. Both human and mouse MTF-1 when produced in transfected mammalian cells strongly bind to a consensus MRE of metallothionein promoters. However, human MTF-1 is more effective than the mouse MTF-1 clone in mediating zinc-induced transcription.

Amino Acid Sequence↗
PubMed1994

Molecular genetic analyses of the Tyrolean Ice Man.

An approximately 5000-year-old mummified human body was recently found in the Tyrolean Alps. The DNA from tissue samples of this Late Neolithic individual, the so-called "Ice Man," has been extracted and analyzed. The number of DNA molecules surviving in the tissue was on the order of 10 genome equivalents per gram of tissue, which meant the only multi-copy sequences could be analyzed. The degradation of the DNA made the enzymatic amplification of mitochondrial DNA fragments of more than 100 to 200 base pairs difficult. One DNA sequence of a hypervariable segment of the mitochondrial control region was determined independently in two different laboratories from internal samples of the body. This sequence showed that the mitochondrial type of the Ice Man fits into the genetic variation of contemporary Europeans and that it was most closely related to mitochondrial types determined from central and northern European populations.

Animals↗
PubMed1994

The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression.

We have described and cloned previously a factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in the enhancer/promoter region of metallothionein genes. MTF-1 is a protein of 72.5 kDa that contains six zinc fingers and multiple domains for transcriptional activation. Here we report the disruption of both alleles of the MTF-1 gene in mouse embryonic stem cells by homologous recombination. The resulting null mutant cell line fails to produce detectable amounts of MTF-1. Moreover, due to the loss of MTF-1, the endogenous metallothionein I and II genes are silent, indicating that MTF-1 is required for both their basal and zinc-induced transcription. In addition to zinc, other heavy metals, including cadmium, copper, nickel and lead, also fail to activate metal-responsive promoters in null mutant cells. However, cotransfection of an MTF-1 expression vector and metal-responsive reporter genes yields strong basal transcription that can be further boosted by zinc treatment of cells. These results demonstrate that MTF-1 is essential for metallothionein gene regulation. Finally, we present evidence that MTF-1 itself is a zinc sensor, which exhibits increased DNA binding activity upon zinc treatment.

Animals↗
PubMed1994

Transcriptional activation modulated by homopolymeric glutamine and proline stretches.

Many transcription factors contain proline- or glutamine-rich activation domains. Here it is shown that simple homopolymeric stretches of these amino acids can activate transcription when fused to the DNA binding domain of GAL4 factor. In vitro, activity increased with polymer length, whereas in cell transfection assays maximal activity was achieved by 10 to 30 glutamines or about 10 prolines. Similar results were obtained when glutamine stretches were placed within a [GAL4]-VP16 chimeric protein. Because these stretches are encoded by rapidly evolving triplet repeats (microsatellites), they may be the main cause for modulation of transcription factor activity and thus result in subtle or overt genomic effects.

Amino Acid Sequence↗
PubMed1994

Functional differences between mammalian transcription activation domains at the yeast GAL1 promoter.

We have fused representatives of three structurally and functionally distinct classes of mammalian transcription activation domains for RNA polymerase II to the yeast GAL4 DNA binding domain. All fusion proteins were stable when expressed in yeast and were tested for their ability to activate transcription from various positions in the yeast GAL1 promoter. Activation domains functional from remote as well as TATA-proximal positions in mammalian cells, e.g. the acidic-type domain of VP16, also stimulate transcription in yeast from various promoter positions. Proline-rich domains, as e.g. in AP-2 and CTF/NF1, with considerable promoter activity and low enhancer activity in mammalian cells stimulate transcription in yeast only from a position close to the TATA box. The glutamine-rich domains of Oct1, Oct2 and Sp1, which activate transcription in mammalian cells from close to the TATA box in response to a remote enhancer, are inactive in the yeast GAL1 promoter. This finding might reflect some basic difference between the organization of yeast and mammalian promoters.

Animals↗
PubMed1994

Basal components of the transcription apparatus (RNA polymerase II, TATA-binding protein) contain activation domains: is the repetitive C-terminal domain (CTD) of RNA polymerase II a "portable enhancer domain"?

Regions rich in serine, threonine, and proline residues can be found in transcriptional activation domains, as well as in the N-terminal parts of mammalian TATA-binding proteins, where they are interrupted by polyglutamine stretches. Likewise, the C-terminal domain of the largest subunit of RNA polymerase II contains multiple repeats of the consensus heptapeptide sequence YSPTSPS. To test directly for possible activation functions, we fused the GAL4 DNA-binding domain to the N-terminal domain of human TBP or subdomains of it, and to the C-terminal domain (CTD) of mouse RNA polymerase II or synthetic polymers of a CTD consensus repeat. We found that these chimeric proteins were able to activate transcription when bound to a GAL4 site in front of the TATA box, a function characteristic of transcription factors. However, while subdomains of TBP functioned only from a position close to the TATA box ("promoter" position), multiple repeats of the CTD consensus sequence were also able to mediate transcriptional activation from a remote ("enhancer") position. Our findings suggest that a region of TBP that is unique to mammals functionally cooperates with "proximal" activation domains of promoter-bound transcription factors. They also imply that the C-terminal domain of RNA polymerase II includes a function that is otherwise confined to remote activation domains of enhancer-bound transcription factors. We suggest that the CTD of RNA polymerase II contains a "portable" remote activation domain that may also facilitate chromatin opening within the transcription unit.

Amino Acid Sequence↗
PubMed1994

Nosocomial transmission of Salmonella gastroenteritis to laundry workers in a nursing home.

BACKGROUND: Outbreaks of salmonella gastroenteritis in nursing homes are common. Person-to-person transmission to nursing home personnel occurs occasionally, but infection of laundry staff as a result of handling soiled linen rarely has been reported. OBJECTIVE: To examine the nosocomial transmission of infection to laundry staff during an outbreak of salmonellosis in a nursing home. SETTING: A 250-bed nursing home in a rural Tennessee county. METHODS: Residents and staff of the nursing home were interviewed and cultures of stool samples examined for enteric pathogens. RESULTS: Stool cultures from 32 residents and 8 employees were positive for Salmonella hadar. Infection among the residents was food-borne, but infection among employees likely represented secondary transmission, as none of the employees ate food prepared in the kitchen and their onset of symptoms occurred seven to 10 days after that of ill residents. Three laundry personnel who had no contact with residents were infected. Most of the ill residents (81%) were incontinent, which led to an increase in both the degree of fecal soiling and the amount of soiled linen received by the laundry during the outbreak. Laundry personnel regularly ate in the laundry room, did not wear protective clothing, and did not wear gloves consistently while handling soiled laundry. CONCLUSIONS: This investigation implicates linen soiled with feces as the source of nosocomial S hadar infection in laundry workers and underscores the importance of using appropriate precautions when handling linen.

Adult↗
PubMed1994

Sustained transmission of mumps in a highly vaccinated population: assessment of primary vaccine failure and waning vaccine-induced immunity.

From January to July 1991, an outbreak of mumps occurred in Maury County, Tennessee. At the primarily affected high school, where 98% of students and all but 1 student with mumps had been vaccinated before the outbreak, 68 mumps cases occurred among 1116 students (attack rate, 6.1%). Students vaccinated before 1988 (the first year mumps vaccination was required for school attendance in Tennessee) may have been at greater risk of mumps than those vaccinated later (65[6.1%] of 1001 vs. 2[2.2%] of 89; risk ratio, 2.9; 95% confidence interval, 0.7-11.6). Of 13 persons with confirmed mumps who underwent serologic testing, 3 lacked IgM antibody in well-timed acute- and convalescent-phase serum specimens. Vaccine failure accounted for a sustained mumps outbreak in a highly vaccinated population. Most mumps cases were attributable to primary vaccine failure. It is possible that waning vaccine-induced immunity also played a role.

Adolescent↗
PubMed1994

Short introns interrupting the Oct-2 POU domain may prevent recombination between POU family genes without interfering with potential POU domain 'shuffling' in evolution.

Transcription factors are often encoded by gene families that share the same type of DNA binding domain. The POU domain genes are one such paradigm. We compared the genomic DNA encoding the POU domain of the Oct-2 genes in human and mouse. In both species this domain is split into a cluster of four exons by short, highly diverged introns. We postulate that the main role of these introns is to prevent ectopic homologous recombination with other members of the POU gene family, with its potentially deleterious effects in somatic and germline cells. Such rapidly diverging introns may generally promote evolution by facilitating the maintenance of duplicated genes. The use of different codons for the same protein domain among members of a gene family may be a slower process that serves a similar purpose. Introns that split conserved domains such as the POU domain do not conform to the exon shuffling hypothesis originally put forward by W. Gilbert (1978). However, we note that the introns flanking the POU domain are in the same phase, i.e. interrupt codons in the same reading frame. Thus we propose that the entire POU domain, which is encoded by a tight cluster of exons, has been shuffled together during evolution as a functional unit, or 'shufflon'.

Animals↗
PubMed1994

Different potential of cellular and viral activators of transcription revealed in oocytes and early embryos of Xenopus laevis.

Many protein domains for transcriptional activation also function when fused to a heterologous DNA binding domain. In mammalian/HeLa cells, we have previously characterized the activation domains of several transcription factors using GAL4 fusion proteins. Here we have tested their transcriptional activity in oocytes and developing embryos of the clawed toad Xenopus laevis. We find that the "acidic" C-terminal domain of the herpesvirus VP16 (= Vmw65) activator, which is active from yeast to man, is also very active in the two Xenopus systems. The constitutive nature of this viral domain may have evolved to be refractory to cellular defense mechanisms. By contrast, activation domains from cellular eukaryotic transcription factors (TFE3, ITF2, MTF-1) are differentially active in oocytes and early embryos. This indicates that their activity can be regulated by protein modification and/or availability of specific coactivators. We have also compared VP16 induced enhancement of transcription from remote and promoter-proximal positions. In both oocytes and late blastula embryos, activation from a promoter-proximal position was more than 50 fold, while only a moderate stimulation (3-8 fold) was observed from remote positions. This may mean that frog oocyte and early embryos are not yet fully geared for gene control by remote enhancers, i.e. respond predominantly to close-by regulatory sequences. The fact that cellular enhancers are naturally located at various distances from the responsive promoters may thus be exploited by multicellular organisms for differential gene control at early and late stages of development.

Animals↗
PubMed1994

A minimal transcription activation domain consisting of a specific array of aspartic acid and leucine residues.

Transcriptional activation by the herpesvirus protein VP16 (= Vmw65, alpha TIF) is mediated by its C-terminal acidic activation domain. Using GAL4 fusion proteins, we have previously shown that a construct containing two tandem copies of a short eleven amino acid fragment derived from the VP16 domain (DALDDFDLDML, residues 437-447) activates transcription in mammalian cells with an efficiency comparable to a GAL4 fusion with the full VP16 activation domain (residues 413-490). Here we have mutagenized this eleven amino acid core sequence and find that a mutant sequence with little inherent activity can cooperate with a wildtype sequence to yield almost full activity. Moreover, greater activity is observed when the wildtype sequence is positioned at the distal, rather than the proximal, end of the fusion protein, indicating that the distal position facilitates contacts to the transcription apparatus. We have also further reduced the eleven amino acid activating sequence to shorter sequence motifs. Two copies of eight and seven amino acids (DALDDFDL and DDFDLDL, respectively), or four copies of the sequence motif DDFDL are required to reach the activation potential of two eleven amino acid motifs. Four copies of the sequence DDLDL still activate transcription strongly (up to two-thirds of DDFDL), indicating that an aromatic residue is not an essential feature of this type of activation domain. However, repetitions of DDL or DL do not yield activity. Thus the minimal requirement for transcriptional activation is the presence of a sequence of some fifteen to twenty amino acids consisting of a specific array of aspartic acid and leucine residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Mitochondrial DNA sequences from Switzerland reveal striking homogeneity of European populations.

Mitochondrial DNA sequences from 74 Swiss individuals were compared to sequences from British and Finish populations. We found that the nucleotide sequence differences between these populations are almost as low as those within the populations. This is in contrast to three African populations, which display substantial differences between each other. The homogeneity of the mitochondrial gene pool in Europe suggests a recent common ancestry for European populations. This may reflect the arrival of anatomically modern humans about 40,000-30,000 years ago or, alternatively, the spread of agriculturalists about 10,000-6,000 years ago. Taking into account the estimated rate of evolution of the mitochondrial control region, the data favor the former explanation.

Black People↗