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K Pfeifer

Publications and source records attributed to K Pfeifer.

At least 55 records · Page 3Linked to original sources
PubMed1991

Evidence for a direct interaction of Rev protein with nuclear envelop mRNA-translocation system.

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of approximately 1 x 10(10) M-1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 125I-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPase-driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclear-envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.

Adenosine Triphosphate↗
PubMed1990

Characterization of the autoantigen La as a nucleic acid-dependent ATPase/dATPase with melting properties.

The autoantigen La, a known transcription termination factor of RNA polymerase III, was purified to homogeneity from mouse 3T3 cells and calf thymus by different isolation procedures. The La protein from calf thymus was separated into RNA binding and nonbinding subclasses. The murine La protein and the RNA binding subclass of calf thymus La protein showed ATPase/dATPase activity in the presence of DNA-RNA or RNA-RNA hybrids. A novel monoclonal anti-La antibody (La11G7) and patients' anti-La antibodies immunoadsorbed to homogeneously purified La protein were able to inhibit the enzyme activity of La protein. La protein was able to melt a synthetic DNA-RNA hybrid in a reaction that required ATP hydrolysis. The RNA binding ability of the nonbinding subclass was restored by treatment with sialidase. This treatment also restored the protein's ATP-dependent melting activity.

Adenosine Triphosphatases↗
PubMed1990

Internal deletions in the yeast transcriptional activator HAP1 have opposite effects at two sequence elements.

In this report we study the effects of internal deletions of the yeast transcriptional activator HAP1 (CYP1) on activity at two dissimilar DNA binding sites, upstream activation sequence 1 (UAS1) of CYC1 (iso-1-cytochrome c) and CYC7 (iso-2-cytochrome c). These deletions remove up to 1061 amino acids of the 1483-residue protein and bring the carboxyl-terminal acidic activation domain closer to the amino-terminal DNA-binding domain. Surprisingly, the deletions have opposite effects at the two sites; activity at UAS1 increases with deletion size, while activity at CYC7 decreases. The mutant with the largest deletion, mini-HAP1, has no measurable activity at CYC7 but binds normally to the site in vitro. In contrast, a protein with the DNA-binding domain of HAP1 fused to the acidic activation domain of GAL4 is active at both UAS1 and CYC7. These findings are discussed in the context of two models that suggest how the DNA sequence can alter the activity of the bound HAP1. In a separate experiment, we generate a mutation in the DNA-binding domain of HAP1 that requires the addition of zinc for binding to either UAS1 or CYC7 in vitro. This finding shows that a zinc finger anchors DNA binding to both types of HAP1 sites.

Chromosome Deletion↗
PubMed1989

The La antigen shuttles between the nucleus and the cytoplasm in CV-1 cells.

Recently we established a monoclonal antibody against the La-protein (Bachmann et al., Proc. Natl. Acad. Sci. USA, 83, 7770, 1986). The antibody gives a nuclear speckled type staining and, in addition, a perinuclear cytoplasmic staining on cultured cells in immunofluorescence microscopy. After inhibition of RNA synthesis the La-protein is transported into the cytoplasm. After prolonged inhibition it returns into the nucleus forming large growing speckles. The transport into the nucleus apparently depends on glycosylation.

Animals↗
PubMed1989

Functional dissection and sequence of yeast HAP1 activator.

We present the DNA sequence and a functional dissection of the 1483 residue yeast activator HAP1. Salient results are, first, a single DNA binding domain (1-148) specifies binding to the two target sites of different sequence, UAS1 and CYC7. This domain contains a cysteine-rich zinc finger, and mutation of either of two cysteines abolishes binding to both sites. Second, mutations that specifically abolish binding to UAS1 or to CYC7 exist. These changes lie either in the residue immediately amino-terminal to the finger or in sequences carboxyl to the finger. Thus, both the base of the finger and carboxyl flanking residues are involved in specific DNA binding. Third, a distinct region (residues 245-445) mediates heme induction by masking the DNA binding domain in the absence of inducer; heme counteracts this masking, perhaps by interacting with a repeat sequence of metal binding character in this region. While sequences between 445 and 1308 have no obvious function, a highly acidic carboxyl terminus mediates transcriptional activation by HAP1.

Amino Acid Sequence↗
PubMed1989

Demonstration of an endocrine signaling circuit for insulin in the sponge Geodia cydonium.

The existence of an insulin-mediated cell-to-cell signaling in the sponge Geodia cydonium is demonstrated in this study by molecular biological and immunological techniques. The sequence of a sponge cDNA clone encoding preproinsulin was analyzed for the first time and determined to comprise a high homology to human preproinsulin (60-80% homology). The predicted polypeptide of preproinsulin from sponge contains two disulfide bridges which link the A- to the B-chain. The intra-A chain disulfide bridge is absent. Applying immunological and electron microscopical techniques it is shown that insulin is produced in specialized cells (spherulous cells). Experimental evidence is presented which indicates that the sponge preproinsulin (predicted Mr 11,850) is processed to insulin (Mr 5600; B-chain, Mr 3700 and A-chain, Mr 1900). Plasma membranes of sponge cells are shown to be provided with an insulin-binding receptor composed of two molecules (Mr 104,000 and Mr 98,000). Heterologous insulin (from bovine pancreas) was found to stimulate gene expression in G. cydonium cells. It is concluded that sponges are provided with an endocrine signaling circuit: signaling cells (spherulous cells), hormone (insulin), and hormone receptor bearing target cells which respond to the hormone stimulus.

Amino Acid Sequence↗
PubMed1989

Identification of La ribonucleoproteins as a component of interchromatin granules.

Monoclonal antibodies raised against the La antigen were used to localize by preembedding immunoelectron microscopy, snRNPs containing this protein. The results demonstrate that La RNPs are localized in clusters of interchromatin granules, both in Triton X-100-extracted and DNase-digested nuclei. DNase-digested salt-extracted nuclei contained, in addition, labeled structures identified as perichromatin granules and fibers. A close association of labeled granules with the nucleoli was also observed. Digestion of nuclei with DNase yielded residual scaffolds of intermediate filaments and nuclear lamina devoid of interchromatin granules and La immunostaining. Release of the La antigen was tested in the presence of ATP and cytochalasin B. Only cytochalasin was effective suggesting a role for nuclear actin in anchorage of snRNPs.

Antibodies, Monoclonal↗
PubMed1988

A monoclonal Ro-antibody and the serum of a Ro-positive patient with subacute cutaneous lupus erythematosus (SCLE) react with basal layers of human epidermis.

Skin lesions, especially at areas exposed to sunlight, prove to be a major form of manifestation of diseases related to Ro-antibodies and neonatal-, 'ANA-negative-', and cutaneous types of lupus erythematosus. A monoclonal Ro-antibody established by our group reacts with a 60 kD polypeptide in extracts from human spleen, whereas in extracts from human epidermis the monoclonal Ro-antibody and a purified Ro-antibody from a monospecific serum of a patient with subacute cutaneous lupus erythematosus reacted with a 60 kD and a 48 kD protein. Performing immunofluorescence microscopy on HEp2-cells both antibodies showed a nuclear speckled staining pattern and a reaction with cytokeratin filaments. On skin biopsies obtained from the patient, predominantly the basal layers of the proliferative stratum germinativum demonstrated a high rate of immunofluorescence; antigen-expression seems to depend on sunlight exposure. From these data we assume that, especially in subacute cutaneous lupus erythematosus, cross-reaction of the Ro-antibody with one of the lower molecular keratins happens in that area of human epidermis which histologically shows cellular destruction.

Antibodies, Antinuclear↗
PubMed1987

Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene.

We show that the yeast HAP1 activator locus encodes a protein that binds in vitro to the upstream activation site, UAS1, of the CYC1 gene (iso-1-cytochrome c). Binding of wild-type HAP1 and truncated HAP1 derivatives to UAS1 is evident in crudely fractionated yeast extracts using the gel electrophoresis DNA binding assay. The binding of HAP1 in vitro, like the activity of UAS1 in vivo, is stimulated by heme. HAP1 binds to region B, one of two portions of UAS1 shown to be important by genetic analysis of the site. Surprisingly, HAP1 binds to the same sequence as a second factor, RC2. Both HAP1 and RC2 bind to the same side of the helix, and make similar but not identical major and minor groove contacts that span two full turns. An additional factor that binds to the second important part of UAS1, the region A factor (RAF), is also identified. A model depicting the interplay of HAP1, RC2, and RAF in the control of UAS1 is presented.

Base Sequence↗
PubMed1987

Yeast HAP1 activator binds to two upstream activation sites of different sequence.

We show that the HAP1 protein binds in vitro to the upstream activation site (UAS) of the yeast CYC7 gene. Strikingly, this sequence bears no obvious similarity to the sequence bound by HAP1 at UAS1 of the CYC1 gene. The CYC1 and CYC7 sites compete for binding to HAP1 and have comparable affinities for the protein. The gross features of the interaction of HAP1 with the two sites are similar: multiple major and minor groove contacts, spanning 23 bp, on one helical face, with a back-side major groove contact toward one end. The precise positions of the contacts differ, however. A mutant form of HAP1, HAP1-18, abolishes the ability of the protein to bind to UAS1 but not CYC7 DNA. Possible mechanisms for how a single protein recognizes two sequences are discussed.

Base Sequence↗
PubMed1987

Identification of the Ro and La antigens in the endoribonuclease VII--ribonucleoprotein complex.

45 S RNP (ribonucleoprotein) particles from calf thymus or L5178y mouse lymphoma cells contain the poly(A)-modulated and oligo(U)-binding endoribonuclease VII [Bachmann, Zahn & Müller (1983) J. Biol. Chem. 258, 7033-7040]. From these particles a 4.5 S RNA was isolated that possesses an oligo(U) sequence. By using monospecific and non-cross-reacting antibodies directed against the La or Ro antigen, both proteins were identified in the endoribonuclease VII-RNP complex after phosphorylation in vitro. In a second approach, endoribonuclease VII activity was identified in immunoaffinity-purified Ro RNPs after preparative isoelectric focusing. Therefore we conclude that the 4.5 S RNA belongs to the Ro RNAs. The results indicate a possible function of endoribonuclease VII in activating stored mRNAs.

Animals↗
PubMed1987

Organization of the regulatory region of the yeast CYC7 gene: multiple factors are involved in regulation.

Regulation of the CYC7 gene of Saccharomyces cerevisiae, encoding iso-2-cytochrome c, was studied. Expression was induced about 20-fold by heme and derepressed 4- to 8-fold by a shift from glucose medium to one containing a nonfermentable carbon source. Deletion analysis showed that induction by heme depends upon sequences between -250 and -228 (from the coding sequence) and upon the HAP1 activator gene, previously shown to be required for CYC1 expression (L. Guarente et al., Cell 36:503-511, 1984). Thus, HAP1 coordinates expression of CYC7 and CYC1, the two genes encoding isologs of cytochrome c in S. cerevisiae. HAP1-18, a mutant allele of HAP1, which increased CYC7 expression more than 10-fold, also acted through sequences between -250 and -228. In vitro binding studies showed that the HAP1 product binds to these sequences (see also K. Pfeifer, T. Prezant, and L. Guarente, Cell 49:19-28, 1987) and an additional factor binds to distal sequences that lie between -201 and -165. This latter site augmented CYC7 expression in vivo. Derepression of CYC7 expression in a medium containing nonfermentable carbon sources depended upon sequences between -354 and -295. The interplay of these multiple sites and the factors that bind to them are discussed.

Chromosome Mapping↗
PubMed1987

Age-correlated changes in the lupus erythematosus antigens Ro, La, Sm and RNP of the thymus gland.

The concentrations of the Sm, RNP, La and Ro antigens of thymus glands from rats were determined depending on the developmental stage of the animals. It was found that lupus antigens strongly decrease after birth. Parallel with this change, the activities of the enzymes DNA polymerase alpha and terminal nucleotidyl transferase in the thymus glands drop during maturation and ageing. These biochemical analyses were supported by immunofluorescence studies using human thymus glands. Moreover, it is documented that a redistribution of Sm and Ro occurs during development. Focusing on Sm, fetal thymus glands contain this antigen predominantly in the cytoplasm, while in immature, mature or old animals Sm is found almost exclusively in the nucleus. From these data we conclude that the amounts of the lupus antigens are additional parameters for the age-correlated function of thymocytes.

Aging↗
PubMed1986

Two or more copies of Drosophila heat shock consensus sequence serve to activate transcription in yeast.

A synthetic oligonucleotide bearing the Drosophila heat shock consensus sequence confers heat inducibility on a CYC1-lacZ gene in Saccharomyces cerevisiae. This sequence CTGGAATTTTCTAGA was inserted in place of the upstream activation sites of the CYC1 promoter adjacent to CYC1 TATA boxes. These constructs were transformed into yeast and found to be heat-inducible when two or more inserts were present. The level of inducibility seemed to increase with the number of inserted sequences: however, the orientations of these sequences relative to each other did not have much effect.

Animals↗
PubMed1986

Association of La and Ro antigens with intracellular structures in HEp-2 carcinoma cells.

Monoclonal antibodies were raised against homogeneous Ro and La antigens, two proteins associated with Ro and La ribonucleoproteins (RNPs). The specificity of the monoclonal antibodies was proven by immunoblot analysis and by immunoprecipitation. The anti-Ro antibody reacted with a Mr 95,000 protein in a mouse lymphoma cell extract and with a Mr 60,000 polypeptide in extracts from human spleen. The anti-La antibody recognized a Mr 50,000 polypeptide in the mouse L5178y cell extract. The two monoclonal antibodies precipitated RNPs that contained the typical RNA species of Ro or La RNPs. The localization of Ro and La antigen was performed by direct immunofluorescence microscopy. It was found that the anti-Ro antibody reacted with a fibrous network that behaves like cytokeratin, one of the intermediate filament systems. The anti-La antibody reacted with nuclear structures that gave a speckled-type pattern.

Animals↗
PubMed1986

Occurrence of novel small RNAs with concomitant inhibition of host cellular U small nuclear RNA synthesis in Vero cells infected with herpes simplex virus type 1.

In eukaryotic cells, small nuclear and small cytoplasmic RNAs (sn- or scRNAs) are associated with distinct proteins, forming ribonucleoproteins (snRNPs or scRNPs). In the present study we analysed the protein composition as well as the small RNA pattern in non-infected and herpes simplex virus type 1 (HSV)-infected Vero cells. We found that concomitantly with the shut-off of host cell mRNA synthesis, synthesis of U-snRNAs was stopped. Due to their stability, however, U-snRNAs were still present in cells 36 h after HSV infection. Besides these RNAs, two novel small RNAs which we termed HVR1 and HVR2 were detected in infected cells. On the basis of their relative mobilities in urea gels, the apparent chain lengths of these newly synthesized RNAs were determined to be 255 and 154 nucleotides respectively. The small RNA-binding proteins Sm, RNP, Ro and La were found to increase up to 15-fold after HSV infection. The data presented suggest that new, virus-coded small RNPs are synthesized which might play a role in the maturation and regulation of HSV-coded RNA transcripts.

Animals↗
PubMed1986

Purification and characterization of the Ro and La antigens. Modulation of their binding affinities to poly(U) by phosphorylation and the presence of ATP.

Both the La and the Ro antigen (the latter for the first time) were purified to apparent homogeneity. Ro was found to be a 94 (90)-kDa and La a 50-kDa polypeptide. Both antigens bind to RNA with a high preference for poly(U). The binding hierarchy is U much greater than G greater than A greater than C for La and U much greater than C greater than G greater than A for Ro. Only 15% of the total amount of La or 21% of that of Ro, present in the L5178y cell extract, is able to bind to poly(U), indicating the existence of RNA binding and nonbinding subclasses of La and Ro. The purified antigens were used for the isolation of monospecific antibodies. These antibodies were specific for their respective antigen and did not cross-react. Both the Ro and the La antigen are phosphorylated in vitro by the cytoplasmic protein kinase CII, whereas the nuclear protein kinases NI and NII are unable to phosphorylate the antigens. After phosphorylation or in the presence of ATP the binding affinity of both antigens to poly(U) strongly decreases. The phosphorylation reaction together with the immunoprecipitation by the monospecific antibodies represents a highly sensitive and specific assay which was used during purification and characterization of the Ro and La antigen.

Adenosine Triphosphate↗