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P Abarzúa

Publications and source records attributed to P Abarzúa.

11 recordsLinked to original sources
PubMed1998

Characterization of the chimeric retinoic acid receptor RARalpha/VDR.

The chimeric receptor, RARalpha/VDR, contains the DNA-binding domain of the retinoic acid receptor (RARalpha) and the ligand-binding domain of the vitamin D receptor (VDR). The ligand-binding properties of RARalpha/VDR are equivalent to that of VDR, with an observed Kd for 1alpha,25 dihydroxy-vitamin D3 (D3) of 0.5 nM. In CV-1 cells, both RARalpha and RARalpha/VDR induce comparable levels of ligand-mediated transcriptional activity from the retinoic acid responsive reporter gene, beta(RARE)3-TK-luciferase, in the presence of the ligand predicted from the receptor ligand-binding domain. Two chimeric RAR receptors were constructed which contained the ligand-binding domain of the estrogen receptor (ER): RARalpha/ER and ER/RARalpha/ER. Both RARalpha/ER and ER/RARalpha/ER bind beta-estradiol with high affinity, and are transcriptionally active only from palindromic RAREs (TREpal and/or (TRE3)3). Only RARalpha/VDR matched in kind and degree the functional characteristics of RARalpha: (1) maximally active from the beta(RARE); (2) moderately active from the TREs; (3) inactive from the retinoic X receptor response elements (RXREs) ApoA1 and CRBP II; (4) forms heterodimers with RXRalpha; and (5) binds to the betaRARE. F9 embryonal carcinoma cell lines were generated which express RARalpha/VDR mRNA (F9RARalpha/VDR cells) and compared with F9 wild-type (F9-Wt) cells, which do not express VDR mRNA. Treatment with all-trans retinoic acid (tRA) inhibits cell growth and induces the differentiation morphology in both F9-Wt and F9-RARalpha/VDR cells; whereas, treatment with D3 is similarly effective only for F9-RARalpha/VDR cells. It is concluded RARalpha/VDR is an useful 'tool' to pinpoint, or to augment transcription from RAREs in gene pathways controlled by RAR without inhibiting the retinoid responsiveness of endogenous RARs.

Animals↗
PubMed1996

Restoration of the transcription activation function to mutant p53 in human cancer cells.

The p53 tumor suppressor gene product is a sequence-specific transcription activator frequently mutated in a variety of human malignancies. Typically, tumor-derived p53 missense mutants are defective in DNA binding and this is likely to result in a failure to active p53-regulated genes. Hence, restoring function to mutant p53 represents an attractive target to develop a novel cancer chemotherapeutic agent. We now show that a small chemically modified peptide derived from p53 restores sequence-specific DNA binding to a subset of p53 mutants. Moreover, when microinjected into human colon carcinoma cells this peptide restores the transcription activation function to endogenous mutant p53 protein. This is the first example showing that a small peptide molecule can reverse the effect of several inactivating missense mutations and restore protein function.

Amino Acid Sequence↗
PubMed1995

Microinjection of monoclonal antibody PAb421 into human SW480 colorectal carcinoma cells restores the transcription activation function to mutant p53.

The p53 tumor suppressor is a transcription factor frequently mutated in human malignancies. Tumor-derived p53 missense mutants are defective in sequence-specific DNA binding and fail to activate p53 target genes. mAb PAb421 was shown previously to restore DNA binding to selected p53 mutants in vitro. Here we show that mAb PAb421 when microinjected into human SW480 colorectal carcinoma cells restores the transcription activation function to the resident mutant p53 (arg to his 273, pro to ser 309). Codon 273 is the second most frequent p53 missense mutant found in human tumors. Our results lend support to the concept of restoring wild-type function to mutant p53 as a strategy for cancer therapy.

Antibodies, Monoclonal↗
PubMed1995

Nonradioactive assay for sequence-specific DNA binding proteins.

A nonradioactive functional assay was developed to quantitate DNA binding proteins. The assay was designed to allow the use of 96-well microplates for high sample throughput. We show that the assay can measure sequence-specific DNA binding of purified proteins as well as DNA binding activity present in whole cell extracts. By using a temperature-sensitive DNA binding protein, we demonstrate that DNA binding as measured by this novel assay correlates with the biological function of the protein.

Animals↗
PubMed1994

Distinct binding determinants for 9-cis retinoic acid are located within AF-2 of retinoic acid receptor alpha.

Retinoids exert their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Retinoid receptor activity resides in several regions, including the DNA and ligand binding domains, a dimerization interface, and both a ligand-independent (AF-1) and a ligand-dependent (AF-2) transactivation function. While 9-cis retinoic acid (RA) alone is the cognate ligand for the RXRs, both 9-cis RA and all-trans RA (t-RA) compete for binding with high affinity to the RARs. This latter observation suggested to us that the two isomers may interact with a common binding site. Here we report that RAR alpha has two distinct but overlapping binding sites for 9-cis RA and t-RA. Truncation of a human RAR alpha to 419 amino acids yields a receptor that binds both t-RA and 9-cis RA with high affinity, but truncation to amino acid 404 yields a mutant receptor that binds only t-RA with high affinity. Remarkably, this region also defines a C-terminal boundary for AF-2, as addition of amino acids 405 to 419 restores receptor-mediated gene activity to a truncated human RAR alpha lacking this region. It is interesting to speculate that binding of retinoid stereoisomers to unique sites within an RAR may function with AF-2 to cause differential activation of retinoid-responsive gene pathways.

Amino Acid Sequence↗
PubMed1992

Characterization of a retinoic acid responsive element isolated by whole genome PCR.

We have used whole PCR in an attempt to isolate novel retinoic acid (RA) responsive genes. We cloned several small genomic fragments from total human DNA containing putative retinoic acid responsive elements (RAREs) selected by direct binding to the retinoic acid receptor alpha (RAR alpha). We report here that an oligonucleotide containing a sequence from one of the cloned human DNA fragments, and referred to as alpha 1, functions as an authentic RARE. It is shown that both RAR alpha and RAR beta produced in Cos cells as well as in vitro translated RAR alpha bind directly and sequence-specifically to the alpha 1RARE. By mutational analysis it is demonstrated that the alpha 1RARE consists of an imperfect direct repeat of the estrogen- and thyroid hormone-related AGGTCA half-site motif separated by a 5 bp spacer. The orientation and spacing of the half-site repeats are shown to play a critical role in RAR recognition. When cloned upstream of a TK-Luc reporter, the alpha 1RARE is shown to confer responsiveness to RA in an orientation-independent fashion in F9 and CV-1 cells. The magnitude of the RA response mediated by the alpha 1RARE differed in these cell lines.

Base Sequence↗
PubMed1989

Regulation and patterns of endogenous and exogenous gene expression during differentiation of embryonal carcinoma cells.

Embryonal carcinoma (EC) cells offer an interesting model system for evaluating differentiation because the cells are pluripotent, thus resembling germ cells and embryonic stem cells, and because a number of agents have been defined that are capable of promoting the differentiation of these cells. This chapter examines how EC cells might be triggered to differentiate, with emphasis on retinoic acid because this compound is a potent, naturally occurring inducer that has been studied extensively in this system. The nature of alterations in gene expression during EC cell differentiation is reviewed from the perspective of evaluating whether these changes are likely to be responsible for, or a result of, the differentiation event. Finally, we consider in molecular terms why EC cells, but not their differentiated derivatives, are refractory to the expression of many viral genomes following infection. Based upon these studies, we propose that fundamental changes in gene expression that are observed when differentiation is triggered in EC cells are likely to be due to the disappearance or neutralization of strong repressor elements.

Cell Differentiation↗
PubMed1984

Mutational analysis of primosome assembly sites. II. Role of secondary structure in the formation of active sites.

Based on their activity as effectors for the ATPase activity of Escherichia coli replication factor Y and as templates for primosome-directed DNA synthesis, single-point mutations in the L- and H-strand primosome assembly sites from pBR322 DNA have been grouped into four classes (Abarzúa, P., Soeller, W., and Marians, K. (1984) J. Biol. Chem. 259, 14286-14292). In this report, the effect of various ligands on the characteristic activities of primosome assembly site class II mutants has been examined. Both Mn2+ and spermidine can, at low levels, substitute for Mg2+ in the activation of wild-type sites as effectors for factor Y-catalyzed hydrolysis of ATP. Class II mutant sites characteristically require higher levels of these ligands for activation, suggesting that the specific higher order structure of an active primosome assembly site is maintained through base pairing within the single-stranded DNA sequence. This conclusion is supported by the following. 1) Excess levels of the E. coli single-stranded DNA-binding protein can inactivate wild-type sites at 1 mM Mg2+. Either the addition of NaCl to 80 mM or an increase in the Mg2+ concentration to 5 mM protects against this inactivation. Class II mutant sites, however, cannot be stabilized by 80 mM NaCl at 1 mM Mg2+, and only some class II mutants can be stabilized at 5 mM Mg2+. 2) Active second-site revertants, isolated in vivo and in vitro, of inactive primosome assembly sites containing multiple-base substitutions have mutated to restore lost base pairs in the proposed stem and loop structure of the sites.

Adenosine Triphosphatases↗
PubMed1984

Mutational analysis of primosome assembly sites. I. Distinct classes of mutants in the pBR322 Escherichia coli factor Y DNA effector sequences.

The assembly of the primosome, a multienzyme complex responsible for priming of lagging-strand DNA synthesis in Escherichia coli, occurs on defined regions of DNA. These primosome assembly sites are on the order of 70 nucleotides in length, yet they share little DNA sequence homology. In order to understand the interaction of the primosomal proteins with these sites, the isolation of single-base substitution mutants of the wild-type sequences has been undertaken. The response of 32 of these mutated primosome assembly sites to increasing concentrations of monovalent and divalent cations when they were used as DNA effectors for E. coli replication factor Y-catalyzed ATP hydrolysis and their efficiency as primosome-dependent DNA replication templates have revealed the existence of four distinct classes of mutations in primosome assembly sites. Class I mutations have essentially no effect on the activities elicited by the DNA site; thus, it is likely that they define nonessential or spacer nucleotide residues. Class II mutated DNAs require higher Mg2+ concentrations than the wild-type DNA to be fully activated as factor Y ATPase effectors and cannot be stimulated in the ATPase reaction by monovalent salt at suboptimal levels of Mg2+. The implication of this mutant phenotype on the role of secondary and tertiary DNA structure in determining an active site is examined in the accompanying article (Soeller, W., Abarzúa, P., and Marians, K. J. (1984) J. Biol. Chem. 259, 14293-14300). Class III mutations coinactivate both the ATPase effector and DNA replication template activity of the site, indicating that they probably represent essential contact points between factor Y and the DNA. Class IV mutated DNAs behave in a manner similar to class II mutated DNAs in the ATPase reaction, but have a replication template activity intermediate between that of the class III and class II mutant DNAs. It is possible that these mutant DNAs are deficient in their ability to catalyze, during primosome assembly, a step subsequent to that of factor Y binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed1984

Enzymatic techniques for the isolation of random single-base substitutions in vitro at high frequency.

A general and efficient method has been developed to generate large numbers of single-base substitution mutations simply and rapidly. A unique f1 phage recombinant DNA cloning vector is described, which contains the phi X174 origin of viral strand DNA synthesis and allows one to direct mutagenesis to any specific segment of DNA. Gapped circular DNA is constructed by annealing viral single-stranded circular DNA [ss(c) DNA] with a mixture of linear duplex DNAs that have had their 3'-OH termini processively digested with Escherichia coli exonuclease III under conditions in which the resulting, newly generated 3'-OH termini present in the various hybrid molecules span the region of interest. Base changes are induced by misincorporation of an alpha-thiodeoxynucleoside triphosphate analog onto this primer-template, followed by DNA repair synthesis. The asymmetric segregation of mutants from wild-type sequences is accomplished by double-stranded replicative form DNA----ss(c) DNA synthesis in vitro, initiated from the phi X174 viral strand origin sequence present on the vector DNA. Mutated ss(c) DNA is screened by the dideoxy chain termination method. In one mutagenesis experiment, 21 independent single-base substitutions were isolated in a 72-nucleotide-long target region. DNA sequence analysis showed that all possible base transversions and transitions were represented.

Bacteriophage phi X 174↗