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Biomedical subjects

W Keller

Publications and source records attributed to W Keller.

At least 109 records · Page 6Linked to original sources
PubMed1986

Generation of histone mRNA 3' ends by endonucleolytic cleavage of the pre-mRNA in a snRNP-dependent in vitro reaction.

Incubation of SP6 generated mouse histone H4 mRNA precursors in nuclear extracts of HeLa cells yields processed mRNA species which end on the 3' adenosine of the conserved terminal ACCA sequence not unlike ten different histone mRNAs isolated from sea urchin embryos which end either on the 3' C or A. In the presence of 20 mM EDTA, the cleaved off 3' spacer portions of the RNA transcripts are readily detected, hence the 3' ends of histone mRNAs arise as a consequence of endonucleolytic cleavage(s) of the precursor RNA. The in vitro cleavage reaction is specifically inhibited by human antisera of the Sm-serotype, but not by control sera and can be rescued by the addition of a preparation of partially purified small nuclear RNPs to the antibody-depleted extract. Interestingly, the snRNP preparation is sufficient to elicit 3' processing of pre-mRNA in the absence of added nuclear extract.

Animals↗
PubMed1985

Purification of a protein required for the splicing of pre-mRNA and its separation from the lariat debranching enzyme.

We have used a complementation assay to test for activities required for the splicing of pre-mRNA in vitro. During the hypotonic lysis of HeLa cells, two components are released from the nuclei that specifically stimulate splicing in an extract prepared from washed nuclei. The two activities separate during chromatography on DEAE-Sepharose. One of these activities [splicing factor (SF)2] co-purified through several steps with the lariat debranching enzyme and with a nuclease which degrades the linear portion of lariat RNAs. These enzymes could, however, be separated from SF2 by chromatography on heparin-Sepharose. SF2 fractionates as a single protein with an apparent mol. wt. of 50 000. SF2 is resistant to mild heat treatment and to treatment with micrococcal nuclease, but it is inactivated by N-ethylmaleimide, suggesting that it is a protein which is not associated with an essential RNA component. When SF2 is absent in a complementation assay, the generation of both intermediates and final products of the splicing reaction is completely abolished. Thus, SF2 functions in an early step of the splicing process.

Cell Nucleus↗
PubMed1985

Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences.

We have investigated the early events of pre-mRNA splicing in vitro by sucrose gradient sedimentation analysis. Time course experiments revealed the assembly, in two steps, of a large (50S) pre-mRNA splicing complex, preceded by formation of two other complexes that sediment at approximately 22S and 35S. Pre-mRNA and the intermediates and products of the in vitro splicing reaction cosediment with the 50S complex, while only pre-mRNA is associated with the 22S and 35S complexes. No splicing is observed in the absence of a 50S complex. Formation of the 50S complex requires ATP, whereas formation of the 22S and 35S complexes does not. U-snRNPs are necessary for assembly of the 35S and the 50S complexes but not for assembly of the 22S complex. Analysis with mutant substrate RNAs demonstrated that a polypyrimidine stretch near the 3' splice site and an intact 5' splice site are absolutely required for splicing complex formation.

Adenosine Triphosphate↗
PubMed1984

A transcription enhancer acts in vitro over distances of hundreds of base-pairs on both circular and linear templates but not on chromatin-reconstituted DNA.

We have analyzed the effect of nucleosome formation and of the simian virus (SV40) enhancer on the efficiency of in vitro transcription. In a whole cell extract made from HeLa cells, nucleosome assembly on DNA results in the formation of chromatin-like complexes. However, transcription was detectable only when the DNA templates were partially or totally depleted of nucleosomes. On nucleosome-free templates, when the SV40 enhancer was present upstream from the complete SV40 early or rabbit beta-globin promoters, there was a five- to tenfold stimulation of specific transcription. When present upstream from its homologous promoter, the SV40 enhancer activated SV40 early transcription independently of its orientation with respect to the coding sequence. Point mutations known to impair the SV40 enhancer function in vivo had a similar effect in vitro. The extent of the enhancing effect was the same with linear or circular templates. When the SV40 enhancer was inserted upstream from the rabbit beta-globin gene, the activation of transcription was reduced with increasing distance between the enhancer and beta-globin upstream promoter elements, but was still significant over a distance of more than 400 base-pairs.

Chromatin↗
PubMed1984

The 5' terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors.

We have investigated the role of small nuclear ribonucleoprotein particles (snRNPs) in the in vitro splicing of messenger RNA precursors by a variety of procedures. Removal of the U-type snRNPs from the nuclear extracts of HeLa cells with protein A-Sepharose-coupled human autoimmune antibodies leads to complete loss of splicing activity. The inhibition of splicing can be prevented by saturating the coupled antibodies with purified nucleoplasmic U snRNPs prior to incubation with nuclear extract. We further demonstrate that an intact 5' terminus of U1 snRNA is required for the functioning of U1 snRNP in the splicing reaction. Antibodies directed against the trimethylated cap structure of the U snRNAs inhibit splicing. Upon removal of the first eight nucleotides of the U1 snRNA in the particles by site-directed hydrolysis with ribonuclease H in the presence of a synthetic complementary oligodeoxynucleotide splicing is completely abolished. These results are in strong support of current models suggesting that a base-pairing interaction between the 5' terminus of the U1 snRNA and the 5' splice site of a mRNA precursor is a prerequisite for proper splicing.

Antigen-Antibody Complex↗
PubMed1984

[Value of sonography, computed tomography and angiography in adrenal diagnosis].

Between 1979 and 1982, 47 patients with pathological process of the adrenals were surgically treated after preceding thorough non-evasive angiographic diagnosis (6 phaeochromocytomas, 20 adrenal adenomas, 8 adrenal hyperplasias, 5 adrenal carcinomas, 5 adrenal cysts and 3 adrenal metastases). The most consistently accurate results were obtained via adrenal phlebography with an accuracy of 94%. This was followed by non-evasive computed tomography (87%). Compared with the data given in literature, hormone determination in the adrenal venous blood was less favourable (79%). Selective adrenal arteriography, which was used less often, yielded correct results in 83% of the cases, whereas the number of accurate diagnoses achieved via sonography was lowest with 54%.

Adenoma↗
PubMed1983

Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts.

Using a whole cell extract of HeLa cells, we synthesized unspliced RNAs containing the first two leaders and the first intervening sequence of the adenovirus 2 major late transcription unit. Upon incubation of these pre-mRNAs in reaction mixtures containing a nuclear extract and a postnuclear fraction (S100), removal of the first intervening sequence and concomitant joining of the first leader to the second leader was observed. This splicing reaction requires proteins, Mg2+ ions, and ATP. The S100 fraction alone has no splicing activity but stimulates splicing when added to the nuclear extract. Upon fractionation of the postnuclear S100 by chromatography on ion exchange and gel filtration columns, the stimulatory activity copurifies with small ribonucleoprotein particles.

Adenosine Triphosphate↗
PubMed1983

[Frequency of kidney cyst wall cancer--results of 22,000 ultrasound studies].

In 22 198 sonographic examinations of the epigastric region, the authors identified 759 renal cysts (3.4%) and 165 renal tumours (0.74%). The percentage of incidence of the renal cysts is identical with that found in post-mortem examinations, whereas tumours were distinctly more frequent. Tumours of the cystic wall had been found in 0.5% of all sonographically diagnosed cysts and in 4% of renal cysts examined via fine-needle biopsy. Of the latter cysts (i.e. out of the 4% of cysts diagnosed by fine-needle biopsy), which were associated with tumours, two had been diagnosed as tumour suspects via sonography. Hence, the sonographic absence of abnormal findings in carcinoma of the renal cyst wall is rare. However, cysts diagnosed via sonography which cannot be subjected to fine-needle biopsy, should be rechecked and followed up at regular intervals. If all possible methods of diagnosis are utilised (I.V. urography, sonography, fine-needle biopsy guided by sonography, computed tomography, angiography), successful differential diagnosis of localised pathological conditions of the kidneys can be achieved in 97% of all cases. Patient risk and cost of diagnosis can be kept at a minimum by proceeding in steps as described in the article. Primary surgical exposure of the kidneys in case of a cystic renal process would now appear justified for therapeutic reasons only, not for the purpose of arriving at a diagnosis.

Adenocarcinoma↗
PubMed1981

Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E. coli.

Double-stranded DNA copies of the single-stranded genomic RNA of foot and mouth disease virus have been cloned into the Escherichia coli plasmid pBR322. A restriction map of the viral genome was established and aligned with the biochemical map of foot and mouth disease virus. The coding sequence for structural protein VP1, the major antigen of the virus, was identified and inserted into a plasmid vector where the expression of this sequence is under control of the phage lambda PL promoter. In an appropriate host the synthesis of antigenic polypeptide can be demonstrated by radioimmunoassay.

Antigens, Viral↗
PubMed1981

Transcription and processing of adenoviral RNA by extracts from HeLa cells.

Concentrated extracts from HeLa cells prepared by the procedure of Sugden and Keller [Sugden, B. & Keller, W. (1973) J. Biol. Chem. 248, 3777-3788] can synthesize RNA molecules of discrete lengths in the presence of DNA templates carrying complete viral transcription units. Accurate initiation of transcription was shown to occur at the "major late" promoter and at the promoter for the "early region III" transcription unit of the adenovirus 2 genome. A cloned fragment of adenovirus 2 DNA containing the early region III transcription unit directs the synthesis of discrete RNA species, many of which correspond in length to spliced early region III mRNAs isolated from adenovirus 2-infected HeLa cells. As shown by hybridization/nuclease S2 analysis, RNA splicing and the formation of specific 3' ends are also taking place in vitro.

Adenoviruses, Human↗
PubMed1980

[Stability of the factorial structure of the AMP system during treatment].

The stability of the factorial structure of the AMP system was studied during the course of psychopharmacological treatments (day 0, 5, 10, 20) of 396 patients mainly suffering from endogenous psychoses. The analysis was based on a reduced item pool of 52 items, which occurred on day 20 in at least 10% of the patients. For the 9-factor solution the factorial structure was stable during treatment for the apathetic, the hallucinatory-desintegrative and the retarded-depression syndrome as well as for the hostility syndrome. The other five AMP syndrome showed no stable structure during the treatment. On the other hand the factorial structure of the 2-factor-solution was found to be stable on day 0, 5, 10 and 20 without exception. These results should be considered when using the AMP system for the documentation of syndromes during the course of therapeutic trials.

Adolescent↗