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W Keller

Publications and source records attributed to W Keller.

At least 91 records · Page 5Linked to original sources
PubMed1991

Purification of the cleavage and polyadenylation factor involved in the 3'-processing of messenger RNA precursors.

Polyadenylation of messenger RNA precursors requires the nucleotide sequence AAUAAA and two factors: poly(A) polymerase and a specificity factor termed cleavage and polyadenylation factor (CPF). We have purified CPF from calf thymus and from HeLa cells to near homogeneity. Four polypeptides with molecular masses of 160, 100, 73, and 30 kDa cofractionate with CPF activity. Glycerol gradient centrifugation and gel filtration indicate that these four proteins form one large complex with a sedimentation constant of 12 S, a Stokes radius near 100 A, and a native molecular mass near 500 kDa. Purified CPF binds specifically to an RNA that contains the AAUAAA sequence. Mutation of the AAUAAA sequence inhibits CPF binding as well as polyadenylation. Purified CPF contains only trace amounts of RNA and does not react with antibodies against common epitopes of small nuclear ribonucleoprotein particles. Thus, contrary to previous indications, CPF does not appear to be a small nuclear ribonucleoprotein particle.

Animals↗
PubMed1991

Purification and characterization of poly(A) polymerase from Saccharomyces cerevisiae.

Poly(A) polymerase was purified 22,000-fold to homogeneity from a whole cell extract of Saccharomyces cerevisiae with a yield of 22%. The enzyme is a monomeric polypeptide with a denatured molecular weight of 63,000. Incorporation of labeled ATP into acid-precipitable material by the purified enzyme proceeds faster with manganese than with magnesium ions. Various RNA homopolymers as well as Escherichia coli tRNA or rRNA can serve as primers. An RNA that terminates at the natural poly(A) site of the CYC1 gene is not more efficiently elongated than several nonspecific substrates, indicating the requirement for additional factors to provide specificity. Elongation of the primer is distributive. Covering of a poly(A) primer with poly(A)-binding protein reduces the enzyme's activity more than 10-fold.

Carrier Proteins↗
PubMed1991

Site-directed ribose methylation identifies 2'-OH groups in polyadenylation substrates critical for AAUAAA recognition and poly(A) addition.

The importance of sugar contacts for the sequence-specific recognition that occurs during polyadenylation of mRNAs was investigated with chemically synthesized substrates containing 2'-O-CH3 groups at selected riboses. An RNA (5'-CUGCAAUAAACAAGU-UAA-3') with 2'-O-CH3 ribose at each nucleotide except for the AAUAAA sequence and 3'-terminal adenosine was efficiently polyadenylated in vitro. Methylation of single riboses within AAUAAA inhibited both poly(A) addition and binding of the specificity factor, but the magnitude of inhibition varied greatly at different nucleotides. Nucleotides that showed sensitivity to base substitutions did not necessarily show sensitivity to ribose methylation, and vice versa. The data indicate that the specificity factor interacts with AAUAAA through RNA-protein contacts involving essential recognition of both sugars and bases at different nucleotide positions.

Base Composition↗
PubMed1991

The three-dimensional structure of canine parvovirus and its functional implications.

The three-dimensional atomic structure of a single-stranded DNA virus has been determined. Infectious virions of canine parvovirus contain 60 protein subunits that are predominantly VP-2. The central structural motif of VP-2 has the same topology (an eight-stranded antiparallel beta barrel) as has been found in many other icosahedral viruses but represents only about one-third of the capsid protein. There is a 22 angstrom (A) long protrusion on the threefold axes, a 15 A deep canyon circulating about each of the five cylindrical structures at the fivefold axes, and a 15 A deep depression at the twofold axes. By analogy with rhinoviruses, the canyon may be the site of receptor attachment. Residues related to the antigenic properties of the virus are found on the threefold protrusions. Some of the amino termini of VP-2 run to the exterior in full but not empty virions, which is consistent with the observation that some VP-2 polypeptides in full particles can be cleaved by trypsin. Eleven nucleotides are seen in each of 60 symmetry-related pockets on the interior surface of the capsid and together account for 13 percent of the genome.

Amino Acid Sequence↗
PubMed1991

Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3' processing signal AAUAAA.

Cleavage and polyadenylation factor (CPF) is required for the cleavage as well as for the subsequent polyadenylation reaction during 3' processing of messenger RNA precursors. Here, we have investigated the interaction of CPF and poly(A) polymerase with short RNA substrates. CPF activates poly(A) polymerase to elongate RNA primers carrying the canonical hexamer recognition signal AAUAAA. CPF specifically binds to such RNA as shown by gel mobility shift assays and competition experiments. Upon binding of CPF, two polypeptides of 35 kDa and 160 kDa can be covalently crosslinked to the RNA by irradiation with UV light. These polypeptides may correspond to the smallest and the largest subunit contained in purified CPF fractions. In addition, chemical modification-exclusion experiments demonstrate that CPF interacts directly with the AAUAAA recognition signal in the RNA. The entire hexamer signal is involved in binding of CPF since modification of any of its bases interferes with complex formation.

Base Sequence↗
PubMed1991

Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase.

cDNA clones encoding mammalian poly(A) polymerase were isolated with probes generated by the polymerase chain reaction based on amino acid sequences derived from the purified enzyme. A bovine cDNA clone was obtained encoding a protein of 82 kDa. Expression in Escherichia coli resulted in the appearance of a poly(A) polymerase activity that was dependent on the addition of the purified specificity factor CPF and the presence of the polyadenylation signal AAUAAA in the RNA substrate. The activity copurified with a polypeptide of the expected size. A second class of cDNAs encoded a polypeptide of 43 kDa which was closely related to the N-terminal half of the 82 kDa protein. Northern blots showed two mRNAs of 4.2 and 2.4 kb that probably correspond to the two classes of cDNAs, as well as a third band of 1.3 kb. The sequence of the N-terminal half of bovine poly(A) polymerase is 47% identical with the amino acid sequence of the corresponding part of yeast poly(A) polymerase. Homologies to other proteins are of uncertain significance.

Amino Acid Sequence↗
PubMed1991

Organization and transient expression of the gene for human U11 snRNA.

The nucleotide sequence of U11 small nuclear RNA, a minor U RNA from HeLa cells, was determined. Computer analysis of the sequence (135 residues) predicts two strong hairpin loops which are separated by seventeen nucleotides containing an Sm binding site (AAUUUUUUGG). A synthetic gene was constructed in which the coding region of U11 RNA is under the control of a T7 promoter. This vector can be used to produce U11 RNA in vitro. Southern hybridization and PCR analysis of HeLa genomic DNA suggest that U11 RNA is encoded by a single copy gene, and that at least three genomic regions could be U11 RNA pseudogenes. A HeLa genomic copy of a U11 gene was isolated by inverted PCR. This gene contains the U11 RNA coding sequence and several sequence elements unique for the U RNA genes. These include a Distal Sequence Element (DSE, ATTTGCATA) present between positions -215 and -223 relative to the start of transcription; a Proximal Sequence Element (PSE, TTCACCTTTACCAAAAATG) located between positions -43 and -63; and a 3' box (GTTAGGCGAAATATTA) between positions + 150 and + 166. Transfection of HeLa cells with this gene revealed that it is functioning in vivo and can produce U11 RNA.

Base Sequence↗
PubMed1990

Sequence requirements in different steps of the pre-mRNA splicing reaction: analysis by the RNA modification-exclusion technique.

The stepwise assembly of splicing complexes and the subsequent splicing reaction were analyzed by the RNA modification-exclusion technique, which generates the equivalent of a complete set of point mutations in a single reaction. We found that although the sequences surrounding the 5' splice site, the branch point, and the 3' splice site, including the 3' AG, were required for presplicing complex formation, modified nucleotides at these positions were not completely excluded. The same sequences were required for splicing complex formation; however, modified nucleotides in these sequences were excluded to a much greater extent.

Adenoviruses, Human↗
PubMed1989

Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro.

We have partially purified a poly(A) polymerase (PAP) from HeLa cell nuclear extract which is involved in the 3'-end formation of polyadenylated mRNA. PAP had a molecular weight of approximately 50 to 60 kilodaltons. In the presence of manganese ions, PAP was able to polyadenylate RNA nonspecifically. However, in the presence of magnesium ions PAP required the addition of a cleavage and polyadenylation factor to specifically polyadenylate pre-mRNAs that contain an intact AAUAAA sequence and end at the poly(A) addition site (precleaved RNA substrates). The purified fraction containing PAP was also required in combination with a cleavage and polyadenylation factor and a cleavage factor for the correct cleavage at the poly(A) site of pre-mRNAs. Since the two activities of the PAP fractions, PAP and cleavage activity, could not be separated by extensive purification, we concluded that the two activities are contained in a single component, a PAP that is also required for the specific cleavage preceding the polyadenylation of pre-mRNA.

Cell Extracts↗
PubMed1989

Assessing gender-related differences in achievement orientation using two different achievement scales.

The consistent finding that the achievement scales of the Adjective Check List (ACL) and Edwards Personal Preference Schedule (EPPS) are not correlated has led researchers to question the validity of both of these measures. This article presents evidence indicating that although these measures are independent, they can predict the same performance criteria. Of interest is that the EPPS predicts outcomes for men, whereas the ACL predicts performance for women. A topical examination of each measure's relationships to other scales provides a tentative rationale for these scales' orthogonality that does not call into question the validity of either measure.

Achievement↗
PubMed1989

[Changes in the cardiac dynamics in passive tile to the supine position--impedance cardiography measurements].

Passive tilt is an effective means to manipulate cardiodynamic processes. While there is a large amount of literature available concerning cardiac function with upright tilt, passive tilt into the supine position is poorly investigated. There is evidence that adapting processes to the new position do not show analogy in both conditions. In this study we investigated cardiodynamic changes occurring with passive-graded tilt from the upright position to 20 degrees headdown tilt. Twenty healthy students of both sexes participated in the study. Using impedance cardiography combined with phonocardiography it was possible to acquire stroke volume, heart rate, Heather index, and systolic time intervals. The whole procedure was repeated to test the reliability of impedance data. There was no steady augmentation of stroke volume. Between 90 degrees and 60 degrees stroke volume remained stable and showed only little change between 60 degrees and 30 degrees. Between 30 degrees and the supine position there was a steep increase of stroke volume. Tilting into the upright position apparently has a different effect. The onset of heart rate decline preceded the increase of stroke volume. It is concluded that only little changes of stroke volume/preload are necessary to induce vagal response. In the 30 degrees position both cardiac output and Heather index were minimal; this may be due to decreased sympathetic output. Systolic time intervals showed volume dependancy. Correlations between the first and second part of the study were high for all variables.

Adult↗
PubMed1988

3' cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP.

We have separated and purified three factors from HeLa cell nuclear extracts that together can accurately cleave and polyadenylate pre-mRNAs containing the adenovirus L3 polyadenylation site. One of the factors is a poly(A) polymerase with a molecular weight of approximately 50-60 kd. The second activity is a cleavage factor with a native molecular weight in the range of 70-120 kd. The third component is a factor (cleavage and polyadenylation factor, CPF) that is needed for the cleavage reaction and, in addition, confers specificity to the poly(A) polymerase activity; the native molecular weight of CPF is approximately 200 kd. Poly(A) polymerase together with CPF is sufficient to specifically polyadenylate pre-mRNA substrates that have been precleaved at the poly(A) addition site. In contrast, all three components are required for accurate cleavage and polyadenylation of pre-mRNA substrates. Further purification of CPF by buoyant density centrifugation, ion exchange, and affinity column chromatography or by gel filtration demonstrates that CPF activity resides in a ribonucleoprotein and copurifies with U11 snRNP.

Endoribonucleases↗
PubMed1987

Separation of multiple components of HeLa cell nuclear extracts required for pre-messenger RNA splicing.

Components essential for nuclear pre-messenger RNA splicing have been partially purified from HeLa cell nuclear extracts by chromatography on DEAE-Sepharose, heparin-Sepharose, Mono Q, and Mono S. We have obtained six fractions which, when combined, efficiently splice a synthetic adenovirus 2 major late RNA substrate in vitro. All fractions contain components that support the formation of splicing intermediates (the cleaved 5' exon and the intron-exon 2 lariat). At least one of the fractions also contains an activity that is essential for the second step in the splicing reaction, namely cleavage at the 3' splice site and exon ligation. Two of the fractions are enriched in the major small nuclear ribonucleoprotein particles U1, U2, U4/U6, and U5. They participate in the formation of the splicing complexes which precedes the cleavage and ligation reactions. The remaining four fractions appear to contain protein factors, as suggested by their resistance to micrococcal nuclease.

Chromatography, Gel↗
PubMed1987

Cleavage and polyadenylation of messenger RNA precursors in vitro occurs within large and specific 3' processing complexes.

We have investigated the assembly of complexes associated with in vitro cleavage and polyadenylation of synthetic pre-mRNAs by native gel electrophoresis. Incubation of SP6-generated pre-mRNA containing the adenovirus L3 polyadenylation site in HeLa cell nuclear extract results in the rapid assembly of specific complexes. Formation of these complexes precedes the appearance of cleaved intermediates and polyadenylated products and is dependent on an intact polyadenylation signal within the pre-mRNA. The specific complexes do not form on RNAs with point mutations in the AAUAAA sequence upstream of the L3 polyadenylation site. Furthermore, such mutant RNAs cannot compete for factors involved in the assembly of specific complexes on wild-type pre-mRNA. Upon complex formation a 67-nucleotide region of the L3 pre-mRNA is protected from RNase T1 digestion. This region contains both the upstream AAUAAA signal and the GU-rich downstream sequences. Cleavage and polyadenylation occur within the specific complexes and the processed RNA is subsequently released. We propose that the assembly of specific complexes represents an essential step during pre-mRNA 3' end formation in vitro.

Adenoviruses, Human↗
PubMed1987

Two spliceosomes can form simultaneously and independently on synthetic double-intron messenger RNA precursors.

We have investigated the formation of splicing complexes in vitro on mRNA precursors (pre-mRNAs) containing two introns. Sucrose gradient sedimentation analysis revealed that the double-intron substrate becomes associated with 60S structures, which are larger than the 50S splicing complexes we previously observed with single-intron pre-mRNA precursors. We have demonstrated that the 60S complex represents the assembly of two single splicing complexes on the individual introns by conversion of the 60S double splicing complexes into single 50S spliceosomes by oligodeoxynucleotide directed RNase H cleavage of the double-intron pre-mRNAs within the middle exon. In addition, we have observed by native gel electrophoresis a transient double 'pre-splicing' complex analogous to the 35S 'pre-splicing' complex previously found with single-intron pre-mRNAs. Our results indicate that splicing complexes can form independently and simultaneously on the individual introns of multi-intron pre-mRNAs and that the assembly of these multiple spliceosomes proceeds with the same stepwise pathway observed for single-intron RNAs.

Cell Nucleus↗