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G Schaffner

Publications and source records attributed to G Schaffner.

34 records · Page 2Linked to original sources
PubMed1992

Amphibian oocytes and sphere organelles: are the U snRNA genes amplified?

The sphere organelles (spheres) of Xenopus and other amphibian oocytes are known to contain small nuclear ribonucleoprotein particles (snRNPs) and have been suggested to play a role in snRNP complex assembly. Coupled with the similarities that exist between spheres and nucleoli and the quantitative and kinetic aspects of snRNA synthesis in the Xenopus oocyte, we have investigated whether or not the U snRNA encoding genes are amplified in Xenopus oogenesis, the spheres being possible sites for the location of such extrachromosomal gene copies. By applying a number of quantitative nucleic acid hybridization procedures to both total and fractionated oocyte and somatic DNA, employing both homologous and heterologous U snRNA gene probes and suitable amplification and non-amplification control probes, we show that the U snRNA genes do not undergo any major amplification in Xenopus oogenesis. Therefore, the analogy between the sphere organelles and nucleoli appears to be limited. The role of the spheres and their relationship to other snRNP containing structures, specifically B snurposomes, and the sphere organizer loci remains obscure.

Animals↗
PubMed1991

A simple procedure for the preparation of protected 2'-O-methyl or 2'-O-ethyl ribonucleoside-3'-O-phosphoramidites.

Protected 2'-O-methyl and 2'-O-ethyl ribonucleoside-3'-O-phosphoramidites were prepared via alkylation of the ribonucleosides at an early stage in the synthesis. Utilizing a strategy of minimal protection, the alkylation was performed with unprotected cytidine and adenosine, or with O6-protected guanosine and N3,5'-O-protected uridine using methyl or ethyl iodide and sodium hydride. In subsequent steps, the introduction of standard protective groups for oligonucleotide synthesis and the concomitant separation from 3'-O-alkylated isomers was accomplished. A modification of the phosphitylation procedure permitted facile isolation of the desired phosphoramidites which show high coupling efficiencies in oligomer assembly.

Ethane↗
PubMed1991

Two-step affinity purification of U7 small nuclear ribonucleoprotein particles using complementary biotinylated 2'-O-methyl oligoribonucleotides.

U7 snRNP is a low-abundance small nuclear ribonucleoprotein particle essential for 3' processing of replication-dependent histone pre-mRNA. We have developed a two-step purification of the particle from TB21 mouse mastocytoma cell nuclear extracts, with about a 20% overall yield, using affinity binding to 2'-O-methyl oligoribonucleotides. The purified particle is homogeneous with respect to RNA content. SDS/PAGE of the U7 snRNP proteins revealed a full complement of the standard core proteins (B, DD', E, F, and G) found in the majority of snRNPs. In addition, two U7-specific polypeptides of 14 kDa and 50 kDa were identified. Summation of the molecular masses of the identified components of the U7 particle yields a particle mass of 249 kDa, in approximate agreement with estimates from sucrose gradient sedimentation (261 kDa) and nondenaturing gradient PAGE (217 kDa).

Animals↗
PubMed1991

2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event.

We describe the synthesis of 2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides and demonstrate their utility as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event. These 2'-O-modified compounds were designed to possess the binding affinity of an RNA molecule towards a complementary RNA target with an enhanced stability against nucleases. The 2'-O-methyl and 2'-O-ethyl antisense compounds function as potent inhibitors of the reaction at 1-10 nM, approximately 5-fold more effective than a natural antisense RNA molecule and requiring an approximate 5-fold excess over the target RNA for 80% inhibition of the processing reaction.

Base Sequence↗
PubMed1989

Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro.

A comparative analysis of ribozyme, antisense RNA, and antisense DNA inhibitors of the in vitro small nuclear ribonucleoprotein U7-dependent histone pre-mRNA processing reaction was performed. RNA molecules complementary to the U7 sequence inhibited in vitro processing of histone pre-mRNA at a sixfold excess over U7. Single-stranded DNA complementary to the entire U7 sequence inhibited the reaction at a 60-fold excess over U7, while a short, 18-nucleotide DNA molecule complementary to the 5' end of U7 inhibited the processing reaction at a 600-fold excess. A targeted ribozyme was capable of specifically cleaving the U7 small nuclear ribonucleoprotein in a nuclear extract and inhibited the U7-dependent processing reaction, but in our in vitro system it required a 1,000-fold excess over U7 for complete inhibition of processing.

Animals↗
PubMed1988

Redundancy of information in enhancers as a principle of mammalian transcription control.

In contrast to prokaryotes, in which strong transcriptional signals can be located within very short DNA segments, typical mammalian enhancers are about 200 base-pairs long. We reasoned that a minimal length of enhancer-active DNA is required for a high transcription rate in higher eukaryotes, and that segments from a single enhancer or from different enhancers might be multimerized or combined to satisfy such a requirement. To test this, enhancer fragments from different viruses were joined in a recombinant simian virus 40 (SV40) and screened for efficiency of viral growth. The 48 combinations tested show that the hypothesis is basically correct. For example, two subfunctional heterologous enhancer fragments can together form a functional enhancer. No enhancer shorter than 84 base-pairs could promote SV40 growth, i.e. in no case did we find a short "superstrong" enhancer segment. To test whether multimerization of a short fragment would result in a strong enhancer, we have synthesized a 50 base-pair enhancer segment derived from Herpesvirus saimiri. One to six copies of this oligonucleotide gave an incremental increase in enhancer activity. We propose, therefore, that mammalian gene regulation is based on a redundancy of information that can be provided either by a combination of different DNA sequence elements, or by multiple copies of the same element. Also, the finding of strong and weak enhancers suggests that in most cases an enhancer is permanently required for transcription of a gene, rather than acting in an all-or-none fashion to establish a transcription complex, after which it becomes dispensable.

Animals↗
PubMed1988

Specific contacts between mammalian U7 snRNA and histone precursor RNA are indispensable for the in vitro 3' RNA processing reaction.

We have made a detailed molecular analysis of the reactions leading to the formation of mature 3' ends in mammalian histone mRNAs. Using two analytical protocols we have identified an essential sequence motif in the downstream spacer which is consistently present, albeit in diffuse form, mammalian histone genes. Tampering with this sequence element completely abolishes 3' processing. However, 3' cleavage in vitro, although at a very much reduced rate, can be detected when the conserved hairpin is deleted from histone precursor mRNAs. U7 snRNA, previously shown to be essential for the maturation of sea urchin histone messages, was isolated from murine cells and the sequence was determined. The approximately 63-nucleotide, trimethyl-G-capped, murine U7 snRNA possesses a sequence shown in the sea urchin U7 to be required for Sm-precipitability, and like the sea urchin U7, the 3' end of murine U7 is encased in a hairpin structure. The 5' sequence of murine U7 exhibits extensive sequence complementarity to the conserved downstream motif of the histone precursor. As expected, oligo-nucleotide-directed RNase H cleavage of this portion of murine U7 inhibits the in vitro processing reaction. These experiments identify a set of specific contacts between mammalian U7 and histone precursor RNA which is indispensable for the maturation reaction.

Animals↗
PubMed1988

The -117 mutation in Greek HPFH affects the binding of three nuclear factors to the CCAAT region of the gamma-globin gene.

The Greek form of hereditary persistence of fetal hemoglobin (HPFH) is associated with a point mutation immediately upstream of the distal of the two CCAAT elements of the A gamma-globin gene. Three proteins present in nuclear extracts of erythroleukemia cells bind to this CCAAT region and contact the nucleotide mutated in Greek HPFH. The ubiquitous CCAAT-binding factor CP1 interacts preferentially with the proximal CCAAT sequence. An erythroid cell-specific factor, referred to as NF-E, binds with a higher affinity to the distal CCAAT region and interacts only with sequences flanking the CCAAT motif. The third protein is the vertebrate homologue of the sea urchin CCAAT displacement protein and recognizes sequences in both CCAAT elements and their flanking sequences. While the point mutation in Greek HPFH slightly strengthens the binding of CP1 and the CCAAT displacement protein, the same base change strongly reduces the binding of NF-E to the distal CCAAT region, suggesting a possible role of NF-E in the repression of gamma-globin genes in adult erythroid cells.

Base Sequence↗
PubMed1988

Functional analysis of the sea urchin U7 small nuclear RNA.

U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. We analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The first domain encompasses the 5'-terminal sequences, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing (F. Schaufele, G. M. Gilmartin, W. Bannwarth, and M. L. Birnstiel, Nature [London] 323:777-781, 1986). Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.

Animals↗
PubMed1987

OVEC, a versatile system to study transcription in mammalian cells and cell-free extracts.

We have developed a vector, OVEC ("oligonucleotide vector") to study DNA sequences involved in the regulation of transcription in mammalian cells. This vector is equally suitable for studying expression in vivo after transfection into cells, or for transcription studies in vitro with cell-free extracts. Putative cis-acting DNA segments from enhancers or promoters can be inserted at a position immediately upstream of the TATA box and coding sequence of the rabbit beta-globin gene. A regulatory DNA segment can be tested by itself or in conjunction with an enhancer located either in an adjacent upstream position, or downstream of the beta-globin gene. S1 nuclease mapping can be used to study transcription from circular and linear templates and run-off transcription in vitro is also feasible. Transcripts from a reference globin gene with a small deletion around the transcription initiation site can be measured with the same S1 nuclease probe and thus serve as an internal standard. We demonstrate the usefulness of OVEC by inserting either short oligonucleotides comprising a metal responsive enhancer element, or the SV40 enhancer, directly upstream of the TATA box. Both constructs yield high levels of correctly initiated transcripts in a transient expression assay in HeLa cells. In a HeLa cell nuclear extract the SV40 enhancer stimulates transcription 40-fold.

Animals↗
PubMedDate not supplied

Mutations in tumour suppressor gene p53 in two feline fibrosarcomas.

Exons 5-7 of the tumour suppressor gene p53 were investigated in genomic DNA of tumours of domestic cats. In one fibrosarcoma investigated we observed a mutation GAG-->AAG (glutamic acid-->lysine) in codon 180; in another there was a mutation CGG-->TGG (arginine-->tryptophane) in codon 248.

Animals↗
PubMedDate not supplied

Sequence of an exon of tumour suppressor p53 gene--a comparative study in domestic animals: mutation in a feline solid mammary carcinoma.

Partial sequence determinations were performed on exon 8 of tumour suppressor gene p53 of cattle, sheep, goat, horse and pig. High sequence homology between these species and other species including dog, cat, chicken and man is demonstrated. A mutation CGG-->TGG (arginine-->tryptophan) was detected in a feline solid carcinoma of the mammary gland.

Animals↗
PubMedDate not supplied

Computational simulation of extravehicular activity dynamics during a satellite capture attempt.

A more quantitative approach to the analysis of astronaut extravehicular activity (EVA) tasks is needed because of their increasing complexity, particularly in preparation for the on-orbit assembly of the International Space Station. Existing useful EVA computer analyses produce either high-resolution three-dimensional computer images based on anthropometric representations or empirically derived predictions of astronaut strength based on lean body mass and the position and velocity of body joints but do not provide multibody dynamic analysis of EVA tasks. Our physics-based methodology helps fill the current gap in quantitative analysis of astronaut EVA by providing a multisegment human model and solving the equations of motion in a high-fidelity simulation of the system dynamics. The simulation work described here improves on the realism of previous efforts by including three-dimensional astronaut motion, incorporating joint stops to account for the physiological limits of range of motion, and incorporating use of constraint forces to model interaction with objects. To demonstrate the utility of this approach, the simulation is modeled on an actual EVA task, namely, the attempted capture of a spinning Intelsat VI satellite during STS-49 in May 1992. Repeated capture attempts by an EVA crewmember were unsuccessful because the capture bar could not be held in contact with the satellite long enough for the capture latches to fire and successfully retrieve the satellite.

Biomechanical Phenomena↗