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C S Pikaard

Publications and source records attributed to C S Pikaard.

35 records · Page 2Linked to original sources
PubMed1994

The RNA polymerase I transcription factor UBF is a sequence-tolerant HMG-box protein that can recognize structured nucleic acids.

Upstream Binding Factor (UBF) is important for activation of ribosomal RNA transcription and belongs to a family of proteins containing nucleic acid binding domains, termed HMG-boxes, with similarity to High Mobility Group (HMG) chromosomal proteins. Proteins in this family can be sequence-specific or highly sequence-tolerant binding proteins. We show that Xenopus UBF can be classified among the sequence-tolerant class. Methylation interference assays using enhancer DNA probes failed to reveal any critical nucleotides required for UBF binding. Selection by UBF of optimal binding sites among a population of enhancer oligonucleotides with randomized sequences also failed to reveal any consensus sequence. The minor groove specific drugs chromomycin A3, distamycin A and actinomycin D competed against UBF for enhancer binding, suggesting that UBF, like other HMG-box proteins, probably interacts with the minor groove. UBF also shares with other HMG box proteins the ability to bind synthetic cruciform DNA. However, UBF appears different from other HMG-box proteins in that it can bind both RNA (tRNA) and DNA. The sequence-tolerant nature of UBF-nucleic acid interactions may accommodate the rapid evolution of ribosomal RNA gene sequences.

Animals↗
PubMed1994

Ribosomal gene promoter domains can function as artificial enhancers of RNA polymerase I transcription, supporting a promoter origin for natural enhancers in Xenopus.

Enhancers of RNA polymerase I transcription in higher eukaryotes are repetitive elements within the intergenic spacers of rRNA genes. In Xenopus and mouse, enhancers and the gene promoter bind the activator protein, upstream binding factor, and in Xenopus, enhancers also share sequence similarity with an upstream domain of the promoter. This upstream promoter domain can act as an efficient enhancer when polymerized and cloned adjacent to a ribosomal gene promoter injected into oocytes. A core promoter domain lacking similarity with spacer sequences in Xenopus laevis but analogous to a repeated sequence in Xenopus borealis can also function as an enhancer. These data demonstrate functional relatedness between the promoter and enhancers, supporting the hypothesis that enhancers could have evolved from duplicated promoter domains that bind essential transcription factors. The ability of upstream binding factor to bind enhancers inactivated by mutation suggests that upstream binding factor binding alone cannot explain enhancer function.

Animals↗
PubMed1994

The RNA polymerase I transactivator upstream binding factor requires its dimerization domain and high-mobility-group (HMG) box 1 to bend, wrap, and positively supercoil enhancer DNA.

Upstream binding factor (UBF) is an important transactivator of RNA polymerase I and is a member of a family of proteins that contain nucleic acid binding domains named high-mobility-group (HMG) boxes because of their similarity to HMG chromosomal proteins. UBF is a highly sequence-tolerant DNA-binding protein for which no binding consensus sequence has been identified. Therefore, it has been suggested that UBF may recognize preformed structural features of DNA, a hypothesis supported by UBF's ability to bind synthetic DNA cruciforms, four-way junctions, and even tRNA. We show here that full-length UBF can also bend linear DNA to mediate circularization of probes as small as 102 bp in the presence of DNA ligase. Longer probes in the presence of UBF become positively supercoiled when ligated, suggesting that UBF wraps the DNA in a right-handed direction, opposite the direction of DNA wrapping around a nucleosome. The dimerization domain and HMG box 1 are necessary and sufficient to circularize short probes and supercoil longer probes in the presence of DNA ligase. UBF's sequence tolerance coupled with its ability to bend and wrap DNA makes UBF an unusual eukaryotic transcription factor. However, UBF's ability to bend DNA might explain how upstream and downstream rRNA gene promoter domains interact. UBF-induced DNA wrapping could also be a mechanism by which UBF counteracts histone-mediated gene repression.

Animals↗
PubMed1993

Functional analysis of Arabidopsis thaliana rRNA gene and spacer promoters in vivo and by transient expression.

In eukaryotes, RNA polymerase I transcription is controlled by DNA elements located within the spacers that separate the tandemly arranged rRNA genes. Unlike rRNA coding sequences, the intergenic spacers evolve rapidly and have little sequence similarity even among closely related species. Nonetheless, the arrangement of functional elements, such as spacer promoters and enhancers, is thought to be highly conserved. Here, we identify spacer promoters in the plant Arabidopsis thaliana, thereby demonstrating their existence in both the plant and animal kingdoms. We also use an Arabidopsis transient expression system to perform transcriptional analysis of the ribosomal gene promoter. Spacer promoters share sequence similarity with the gene promoter from -91 to +22 relative to the transcription start site, +1. Deletion analysis shows that sequences required for RNA polymerase I transcription reside within these boundaries. Spacer sequences upstream of the gene promoter have only a small positive effect on transcription in transfected protoplasts but can increase transcription from a Xenopus ribosomal gene promoter in injected frog oocytes. This trans-kingdom enhancer effect further suggests that the functional elements within eukaryotic ribosomal genes are highly conserved.

Animals↗
PubMed1992

Cooperative binding of the Xenopus RNA polymerase I transcription factor xUBF to repetitive ribosomal gene enhancers.

Upstream binding factor (UBF) is a DNA-binding transcription factor implicated in ribosomal gene promoter and enhancer function in vertebrates. UBF is unusual in that it has multiple DNA-binding domains with homology to high-mobility-group (HMG) nonhistone chromosomal proteins 1 and 2. However, a recognizable DNA consensus sequence for UBF binding is lacking. In this study, we have used gel retardation and DNase I footprinting to examine Xenopus UBF (xUBF) binding to Xenopus laevis ribosomal gene enhancers. We show that UBF has a minimum requirement for about 60 bp of DNA, the size of the short enhancer variant in X. laevis. Stronger UBF binding occurs on the longer enhancer variant (81 bp) and on multiple enhancers linked head to tail. In vivo, Xenopus ribosomal gene enhancers exist in blocks of 10 alternating 60- and 81-bp repeats within the intergenic spacer. In vitro, UBF binds cooperatively to probes with 10 enhancers, with five intermediate complexes observed in titration experiments. This suggests that, on average, one UBF dimer binds every two enhancers. A single UBF dimer can produce a DNase I footprint ranging in size from approximately 30 to about 115 bp on enhancer probes of different lengths. This observation is consistent with the hypothesis that multiple DNA-binding domains or subdomains within UBF bind independently, forming more-stable interactions on longer probes.

Animals↗
PubMed1991

xUBF and Rib 1 are both required for formation of a stable polymerase I promoter complex in X. laevis.

We show that three protein fractions are required for accurate transcription initiation at a Xenopus laevis ribosomal gene promoter in vitro: RNA polymerase I, Rib1 and xUBF. The Rib1 and xUBF fractions are both necessary and sufficient for formation of a stable initiation complex. The xUBF fraction can be completely replaced by recombinant xUBF. We also report the sequence of a cDNA clone for xUBF. xUBF is 701 amino acids in length, contains domain which are related to a domain found in chromosomal proteins HMG 1 and 2, and has an acidic carboxy terminus of 87 amino acids. xUBF is closely similar in amino acid sequence to its previously reported human homolog, hUBF, except that xUBF has only three of the HMG-related domains while hUBF has four and therefore is 63 amino acids longer than xUBF.

Amino Acid Sequence↗
PubMed1990

rUBF, an RNA polymerase I transcription factor from rats, produces DNase I footprints identical to those produced by xUBF, its homolog from frogs.

Rat cells contain a DNA-binding polymerase I transcription factor, rUBF, with properties similar to UBF homologs that have been purified from both human (hUBF) and frog (xUBF) cells. In this note we report the affinity purification of rUBF to apparent homogeneity and show that UBFs from both rat and frog have identical footprinting characteristics on templates from either species. Furthermore, xUBF was able to stimulate transcription from rat RNA polymerase I promoters in a partially fractionated rat extract that was UBF dependent. These results strengthen the conclusion that all vertebrate cells contain a UBF homolog whose DNA-binding specificity and function have been strongly conserved.

Animals↗
PubMed1990

Enhancers for RNA polymerase I in mouse ribosomal DNA.

The intergenic spacer of the mouse ribosomal genes contains repetitive 140-base-pair (bp) elements which we show are enhancers for RNA polymerase I transcription analogous to the 60/81-bp repetitive enhancers (enhancers containing a 60-bp and an 81-bp element) previously characterized from Xenopus laevis. In rodent cell transfection assays, the 140-bp repeats stimulated an adjacent mouse polymerase I promoter when located in cis and competed with it when located in trans. Remarkably, in frog oocyte injection assays, the 140-bp repeats enhanced a frog ribosomal gene promoter as strongly as did the homologous 60/81-bp repeats. Mouse 140-bp repeats also competed against frog promoters in trans. The 140-bp repeats bound UBF, a DNA-binding protein we have purified from mouse extracts that is the mouse homolog of polymerase I transcription factors previously isolated from frogs and humans. The DNA-binding properties of UBF are conserved from the mouse to the frog. The same regulatory elements (terminators, gene and spacer promoters, and enhancers) have now been identified in both a mammalian and an amphibian spacer, and they are found in the same relative order. Therefore, this arrangement of elements probably is widespread in nature and has important functional consequences.

Animals↗
PubMed1989

Molecular mechanisms governing species-specific transcription of ribosomal RNA.

An unusual property of ribosomal RNA transcription is the species specificity of promoter recognition. Unexpectedly, the sequence-specific RNA pol I transcription factors hUBF and xUBF, isolated from human and Xenopus cells, respectively, recognize the same DNA sequence elements. Despite this similarity in DNA binding activity, neither factor will functionally substitute for the other in reconstituted transcription assays, suggesting that the specificity of protein-DNA interactions cannot account for the species-specific activation of transcription by hUBF and xUBF. Interestingly, we find that hUBF and xUBF form distinctly different complexes with human SL1 at both the human and Xenopus promoters. Together these results strongly implicate specific protein-protein interactions between transcription factors as an important determinant of promoter selectivity and species specificity.

Animals↗
PubMed1989

The Xenopus ribosomal gene enhancers bind an essential polymerase I transcription factor, xUBF.

We purified xUBF on the basis of its ability to specifically bind the enhancer elements of the Xenopus laevis rRNA genes. xUBF also binds to both upstream and downstream regions of the X. laevis ribosomal gene promoter and is essential for polymerase I transcription. Unexpectedly, xUBF binds to both upstream and downstream regions of the human ribosomal gene promoter, producing footprints that are indistinguishable from the footprints produced by hUBF, a previously described polymerase I transcription factor isolated from human cells. Despite extensive sequence divergence of vertebrate polymerase I promoters, these data suggest an evolutionary conservation of the primary DNA-protein interaction.

Animals↗
PubMed1988

Molecular characterization of the patatin multigene family of potato.

Patatin is a family of glycoproteins that accounts for up to 40% of the total soluble protein in potato tubers. We isolated and characterized 25 patatin genomic clones. All of these exhibit different restriction patterns, but can be divided into two classes based on the presence (class II) or absence (class I) of a 22-bp sequence within the 5'-untranslated region. We determined the complete nucleotide sequence of the class-I clone PS20 and the 5'-flanking sequence of seven additional clones. The transcribed region of PS20 spans 3197 bp and is divided by six introns. The seven exons encode a transcript which is identical to the cDNA, cloned in pGM203 [Mignery et al., Nucl. Acids Res. 12 (1984) 7987-8000]. The 5'-flanking sequences of both class-I and class-II patatin genes are highly homologous up to bp position -87 and then diverge. This conserved region contains the CAAT and TATA homologies as well as a homology to the core enhancer sequence. Within a class there are additional large regions of homology, but differences exist that allow the patatin genes to be further divided into several discrete subclasses. S1 nuclease protection experiments with both class-I and class-II probes showed that class-I transcripts are the predominant species present in tubers. Class-II transcripts are present in tubers, but are 50-100-fold less abundant. In potato roots, class-II transcripts are the predominant species and few, if any, class-I transcripts are present.

Amino Acid Sequence↗
PubMed1988

Sequence elements essential for function of the Xenopus laevis ribosomal DNA enhancers.

The intergenic spacer region of the Xenopus laevis ribosomal DNA contains multiple elements which are either 60 or 81 base pairs long. Clusters of these elements have previously been shown to act as position- and distance-independent enhancers on an RNA polymerase I promoter when located in cis. By a combination of deletion and linker scanner mutagenesis we show that the sequences essential for enhancer function are located within a 56-base-pair region that is present in both the 60- and 81-base-pair repeats. Within the 56-base-pair region one linker scanner mutation was found to be relatively neutral, suggesting that each enhancer element may be composed of two smaller domains. Each 56-base-pair region appears to be an independent enhancer with multiple enhancers being additive in effect. We review the current evidence concerning the mechanism of action of these enhancers.

Animals↗
PubMed1987

The two classes of genes for the major potato tuber protein, patatin, are differentially expressed in tubers and roots.

The major potato tuber protein, patatin, is a family of 40kd glycoproteins that constitutes forty per cent of the soluble protein in tubers but is generally undetectable in other tissues. Fused rocket immunoelectro-phoresis was used to detect in roots patatin that is immunologically different from tuber patatin. Western blots of SDS-polyacrylamide gels show root patatin to have a different molecular weight distribution than tuber patatin isoforms, but immunoprecipitation of in vitro translation products shows the patatin precursors to be of similar molecular weight in both tissues. This suggests that post-translational processing may differ in tubers and roots. Northern blots show that tuber and root patatin mRNAs are of similar size, but tuber transcripts are about 100-fold more abundant. 5' S1 nuclease and primer extension mapping suggests the class of patatin transcripts expressed in roots (class II transcripts) to be a subset of patatin transcripts expressed in tubers (classes I and II). Class II patatin mRNAs differ from class I transcripts by the presence of a 22 nucleotide insertion just upstream of the initiation codon. These data demonstrate that expression of the patatin multigene family is differentially regulated in tubers and roots.

Base Sequence↗
PubMed1984

Isolation and sequence analysis of cDNAs for the major potato tuber protein, patatin.

A cDNA library from membrane-bound poly(A)+ mRNA of developing potato tubers was constructed and two classes of essentially full-length cDNA clones for a precursor to the major tuber glycoprotein, patatin were isolated. Sequence analysis shows that the two classes are approximately 99% homologous and correspond to the two major species of patatin identified previously by NH2-terminal amino acid sequence analysis. Sequence analysis also predicts that patatin is synthesized with a 23 amino acid signal sequence. Northern blot analysis shows that patatin mRNAs are 1550 +/- 50 nucleotides in length and are normally not present in polyribosomal or total RNA from stems or leaves.

Amino Acid Sequence↗
PubMed1984

Maintenance of Normal or Supranormal Protein Accumulation in Developing Ovules of Glycine max L. Merr. during PEG-Induced Water Stress.

Protein accumulation in developing ovules of hydroponically grown soybean (Glycine max L. Merr.) plants was unaffected or enhanced by polyethylene glycol-induced water stress. The cultivar Wayne and the experimental variety ;9656' were severely stressed by inclusion of PEG-6000 in the nutrient solution to water potentials as low as -20 bar. Leaves rapidly yellowed and abscised under these conditions. Fresh and dry weight of ;Wayne' ovules was reduced by severe stress, but protein content was unaffected. Ovules of 9656 were more resistant to severe stress: fresh weight and dry weight were unaffected by stress and protein content increased. Moderately stressed Wayne ovules behaved like severely stressed 9656 ovules: seed fresh weight and dry weight were unaffected and protein content increased. However, protein content did not increase if the plants were defoliated. No changes in seed protein quality were observed with stress, based on polypeptide banding patterns after one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Journal Article↗
PubMed1984

Heat Shock Proteins in Tobacco Cell Suspension during Growth Cycle.

Tobacco (Nicotiana tabacum L. cv Wisconsin 38) cells grown in suspension culture at 26 degrees C produce heat shock proteins (HSPs) when exposed to elevated temperature of 34 to 42 degrees C. At 34 and 38 degrees C, synthesis of normal proteins is maintained while HSPs are expressed within 30 minutes after initiation of the shock. At 42 degrees C, HSPs are still expressed but normal proteins are made at a reduced rate or not at all. Exposure of cells to 38 degrees C allows for a full expression of HSPs without inhibition of the synthesis of normal proteins. Induced synthesis of HSPs at 38 degrees C is maximal 1 to 2 hours after elevation of temperature and diminishes thereafter through at least 6 hours. Cells growing asynchronously in the logarithmic phase of growth produce HSPs at a much higher rate than those in the stationary phase. The ability to synthesize HSPs disappears about one generation time before the cells reach a growth plateau.

Journal Article↗