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M K Darby

Publications and source records attributed to M K Darby.

17 recordsLinked to original sources
PubMed2000

Specific RNA binding by a single C2H2 zinc finger.

Zinc finger proteins with high affinity for human immunodeficiency virus Rev responsive element stem loop IIB (RRE-IIB) were previously isolated from a phage display zinc finger library. Zinc fingers from one of these proteins, RR1, were expressed individually and assayed for RRE-IIB affinity. The C-terminal zinc finger retained much of the binding affinity of the two-finger parent and was disrupted by mutations predicted to narrow the RRE-IIB major groove and which disrupt Rev binding. In contrast, the N-terminal zinc finger has a calculated affinity at least 1000-fold lower. Despite the high affinity and specificity of RR1 for RRE-IIB, binding affinity for a 234-nucleotide human immunodeficiency virus Rev responsive element (RRE234) was significantly lower. Therefore, zinc finger proteins that bind specifically to RRE234 were constructed using an in vitro selection and recombination approach. These zinc fingers bound RRE234 with subnanomolar dissociation constants and bound the isolated RRE-IIB stem loop with an affinity 2 orders of magnitude lower but similar to the affinity of an arginine-rich peptide derived from Rev. These data show that single C2H2 zinc fingers can bind RNA specifically and suggest that their binding to stem loop IIB is similar to that of Rev peptide. However, binding to RRE234 is either different from stem loop IIB binding or the tertiary structure of stem loop IIB is changed within the Rev responsive element.

Amino Acid Sequence↗
PubMed1998

The role of zinc finger linkers in p43 and TFIIIA binding to 5S rRNA and DNA.

Transcription factor IIIA (TFIIIA) and p43 zinc finger protein form distinct complexes with 5S ribosomal RNA in Xenopus oocytes. Additionally, TFIIIA binds the internal promoter of the 5S RNA gene and supports assembly of a transcription initiation complex. Both proteins have nine tandemly repeated zinc fingers with almost identical linker lengths between corresponding fingers, yet p43 has no detectable affinity for the 5S RNA gene. TFIIIA zinc fingers 1-3 are connected by highly conserved linkers, first identified in the Drosophila protein Krüppel, that are found in many DNA binding zinc finger proteins. To understand the role of these linkers in RNA and DNA binding we exchanged three TFIIIA linker amino acids with the equivalent amino acids from p43. The major effect of linker substitution is a 50-fold reduction in DNA specificity, concomitant with an 8-fold reduction in affinity. N-Terminal zinc fingers from either TFIIIA or p43 bind to multiple specific sites on 5S RNA that are resistant to competition by tRNA or poly(rA). This mode of RNA binding is unaffected by linker substitution. These data suggest that zinc finger linkers significantly facilitate the specificity of DNA binding.

Amino Acid Sequence↗
PubMed1998

Specific RNA binding proteins constructed from zinc fingers.

A zinc finger library with degenerate alpha-helices was displayed on the surface of bacteriophage and proteins that bind human immunodeficiency virus type-1 (HIV-1) Rev response element stem loop IIB (RRE-IIB) RNA or 5S rRNA were isolated. DNA encoding affinity selected zinc fingers was shuffled by recombination in vitro to isolate proteins with higher RNA binding affinity. Proteins constructed in this way bind RNA specifically both in vitro and in vivo. These results demonstrate that RNA substrate specificity of zinc fingers can be changed through mutation of alpha-helices to construct novel RNA binding proteins.

Amino Acid Sequence↗
PubMed1997

Phage display of RNA binding zinc fingers from transcription factor IIIA.

Zinc fingers in transcription factor IIIA (TFIIIA) contribute differentially to RNA and DNA binding affinity. We investigated whether the same putative alpha-helix amino acids in TFIIIA zinc fingers are essential for both RNA and DNA binding. In published structures, zinc fingers make DNA base contacts through amino acids -1, +2, +3, and +6 of the recognition helix. Alanine substitution at these four positions were made in TFIIIA RNA binding zinc fingers, tz4-7 and DNA binding zinc fingers, tz1-3. Substitution in zinc fingers 4 or 6 of tz4-7 reduced RNA affinity 77- and 38-fold, respectively, whereas substitution in zinc fingers 5 or 7 had little effect. DNA binding affinity of tz1-3 was eliminated by alanine substitution in any one zinc finger. We determined which amino acids supported RNA binding by phage display of a library of zinc finger 4 mutants. Lysine at helix position -1 of zinc finger 4 was conserved in all selected tz4-7 fusions. Point mutation of Lys-1 to alanine in zinc finger 4 reduced tz4-7 RNA affinity 30-fold. We propose that RNA binding by TFIIIA shows similarity to DNA binding in the use of the recognition helix. Helix positions -1 and +2 may have particular significance for RNA binding.

Amino Acid Sequence↗
PubMed1993

A position-dependent transcription-activating domain in TFIIIA.

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

ATP-Binding Cassette Transporters↗
PubMed1992

Differential binding of zinc fingers from Xenopus TFIIIA and p43 to 5S RNA and the 5S RNA gene.

Zinc fingers are usually associated with proteins that interact with DNA. Yet in two oocyte-specific Xenopus proteins, TFIIA and p43, zinc fingers are used to bind 5S RNA. One of these, TFIIIA, also binds the 5S RNA gene. Both proteins have nine zinc fingers that are nearly identical with respect to size and spacing. We have determined the relative affinities of groups of zinc fingers from TFIIIA for both 5S RNA and the 5S RNA gene. We have also determined the relative affinities of groups of zinc fingers from p43 for 5S RNA. The primary protein regions for RNA and DNA interaction in TFIIIA are located at opposite ends of the molecule. All zinc fingers from TFIIIA participate in binding 5S RNA, but zinc fingers from the C terminus have the highest affinity. N-terminal zinc fingers are essential for binding the 5S RNA gene. In contrast, zinc fingers at the amino terminus of p43 are essential for binding 5S RNA.

Amino Acid Sequence↗
PubMed1990

A finger protein structurally similar to TFIIIA that binds exclusively to 5S RNA in Xenopus.

A 5S RNA binding protein (p43) in Xenopus is a major constituent of oocytes and comprises part of a 42S ribonucleoprotein storage particle. We have cloned and sequenced p43 cDNA from X. laevis and X. borealis as well as the cDNA for X. borealis TFIIIA. Like TFIIIA, p43 has nine zinc fingers, seven of which are exactly the same size as their counterparts in TFIIIA. Amino acid homology between the two proteins is restricted mainly to conserved residues characteristic of zinc fingers. In contrast to TFIIIA, which binds specifically to both 5S RNA and 5S RNA genes, p43 binds exclusively to 5S RNA.

Amino Acid Sequence↗
PubMed1990

The characterization of the TFIIIA synthesized in somatic cells of Xenopus laevis.

In somatic cells of Xenopus, transcription of the TFIIIA gene initiates greater than 200 bp upstream from the start site used in oocytes. The resultant mRNA encodes a protein, S-TFIIIA, that is 22 amino acids longer at its amino terminus than the abundant form of TFIIIA in oocytes (O-TFIIIA). S-TFIIIA binds the 5S RNA gene and 5S RNA, and both O- and S-TFIIIA promote the formation of stable transcription complexes on oocyte-type 5S RNA genes in an oocyte nuclear extract. We have not found any functional difference between the two forms of TFIIIA. Different transcription start sites suggest differential promoter usage--one in oocytes that permits high levels of gene activity and another that is used in somatic cells for low-level TFIIIA mRNA synthesis.

Amino Acid Sequence↗
PubMed1988

Transcription complexes that program Xenopus 5S RNA genes are stable in vivo.

The long-term stability of transcription complexes on 5S RNA genes has been demonstrated in vivo. Complexes on oocyte and somatic-type 5S RNA genes injected into Xenopus laevis oocyte nuclei are stable for at least 4 days. Tissue culture cells and mature erythrocytes have equivalent numbers of somatic 5S RNA genes programmed into transcription complexes, yet the former cell type has a greater than 50-fold higher cellular content of transcription factor IIIA (TFIIIA). Functional transcription complexes on somatic 5S RNA genes in nucleated erythrocytes of Xenopus are stable for weeks, perhaps months, even though a mature erythrocyte has less than two molecules of TFIIIA for each somatic 5S RNA gene. These findings strengthen our proposal that stable transcription complexes are a means of maintaining the differentiated state.

Animals↗
PubMed1988

A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo.

A monoclonal antibody that reacts with proteins in the nuclear pore complex of rat liver (Snow, C. M., A. Senior, and L. Gerace. 1987. J. Cell Biol. 104:1143-1156) has been shown to cross react with similar components in Xenopus oocytes, as determined by immunofluorescence microscopy and immunoblotting. We have microinjected the antibody into oocytes to study the possible role of these polypeptides in nucleocytoplasmic transport. The antibody inhibits import of a large nuclear protein, nucleoplasmin, in a time- and concentration-dependent manner. It also inhibits export of 5S ribosomal RNA and mature tRNA, but has no effect on transcription or intranuclear tRNA processing. The antibody does not affect the rate of diffusion into the nucleus of two small proteins, myoglobin and ovalbumin, indicating that antibody binding does not result in occlusion of the channel for diffusion. This suggests that inhibition of protein and RNA transport occurs by binding of the antibody at or near components of the pore that participate in mediated transport.

Animals↗
PubMed1986

DNA topoisomerase II cleaves at specific sites in the 5' flanking region of c-fos proto-oncogenes in vitro.

We have analyzed 1 kb of cloned human c-fos sequence (-711 to +287) for calf thymus DNA topoisomerase II cleavage sites in vitro. Using the anti-tumor drug VP16 (demethylepipodophyllotoxin-beta-D-glucoside) with purified topoisomerase II, we identify twelve sites. Five sites are clustered around position -306 in a region that possesses enhancer-like properties. A second cluster of three sites is positioned 15 bp upstream of the TATA promoter element. With a HeLa nuclear extract as a source of topoisomerase II, a subset of cleavage sites is conserved within the two clusters. The cleavage sites in the enhancer-like element are conserved in the homologous region of the murine c-fos. These findings raise the possibility that topoisomerase II is involved in mediation of mitogen-induced c-fos expression.

Base Sequence↗
PubMed1985

Relaxation of supercoiled phosphorothioate DNA by mammalian topoisomerases is inhibited in a base-specific manner.

The nucleotide preferences of calf thymus topoisomerases I and II for recognition of supercoiled DNA have been assessed by the relaxation and cleavage of DNA containing base-specific phosphorothioate substitutions in one strand. The type I enzyme is inhibited to varying degrees by all modified DNAs, but most effectively (by approximately 60%) if deoxyguanosine 5'-O-(1-thiomonophosphate) (dGMP alpha S) is incorporated into negatively supercoiled DNA. A DNA in which all internucleotide linkages of one strand are phosphorothionate is relaxed, most probably via the unsubstituted strand. The type II enzyme is inhibited when deoxyadenosine 5'-O-(1-thiomonophosphate) (dAMP alpha S) or deoxyribosylthymine 5'-O-(1-thiomonophosphate) is incorporated into the DNA substrate, and the course of the relaxation reaction changes from a distributive mode to a predominantly processive mode. A fully substituted DNA is very poorly relaxed by the type II enzyme, illustrating the strict commitment of the enzyme to relaxation via double-strand cleavage. The sense of supercoiling does not affect the inhibition profile of either enzyme. DNA strand breaks introduced by type II topoisomerase in a normal control DNA or deoxycytidine 5'-O-(1-thiomonophosphate)-substituted DNA on treatment with sodium dodecyl sulfate at low ionic strength are prevented by pretreatment with 0.2 M NaCl. In contrast, breaks in DNA having either dAMP alpha S or all four phosphorothioate nucleotides incorporated in one strand are prevented only with higher NaCl concentrations. Thus indicating activity at the phosphorothioate linkage 5' to dA but not 5' to dC. We conclude that topoisomerase II activity occurs preferentially at sites possessing dAMP or dTMP, and that dGMP is involved in DNA recognition by topoisomerase I.

Animals↗
PubMed1985

Inhibition of calf thymus type II DNA topoisomerase by poly(ADP-ribosylation).

The effect of poly(ADP-ribosylation) on calf thymus topoisomerase type II reactions has been investigated. Unknotting of phage P4 head DNA, and relaxation and catenation of supercoiled PM2 DNA are inhibited. We conclude that the inhibition results from poly(ADP-ribosylation) on the following grounds. Firstly, the enzyme poly(ADP-ribose) (PADPR) synthetase and NAD are required, secondly, the competitive synthetase inhibitor nicotinamide abolishes topoisomerase inhibition, and thirdly, the polymer alone is not inhibitory. The mechanism of inhibition appears to be disruption of the strand cleavage reaction. A topoisomerase-DNA complex can be formed that upon treatment with protein denaturant at low ionic strength results in strand cleavage. The amount of DNA present in such a cleavable-complex progressively decreased following pretreatment of topoisomerase type II with PADPR synthetase and increasing concentrations of NAD. Treatment of the pre-formed complex with NAD and PADPR synthetase had no effect on its salt-induced dissociation. This suggests that either poly(ADP-ribosylation) has no influence on dissociation of topoisomerase, in contrast to association, or topoisomerase is not accessible to the synthetase when bound to DNA. Similar data were obtained with calf thymus type I topoisomerase.

Animals↗
PubMed1983

Metal-nucleotide structure at the active sites of the mammalian hexokinases.

The diastereomers of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) in the presence of Mg2+, Co2+ and Cd2+ have been used to determine the stereospecificity of the metal-nucleotide binding site of rat muscle hexokinase type II and rat liver glucokinase by the method developed by Jaffe and Cohn [J. Biol. Chem. 254, 10839-10845 (1979)]. The kinetic parameters, Km and V, for the mammalian hexokinase reaction have been determined for ATP beta S in the presence of the three divalent metal ions. In the presence of Mg2+, both enzymes exhibit a preference for the B diastereomer of ATP beta S (V ratio, B/A approximately equal to 20). With Cd+, the stereospecificity is reversed and the A diastereomer is the preferred substrate, suggesting direct coordination of S on the beta-P to this metal ion. Co2+ exhibits a decreased specificity for the B diastereomer over Mg2+. This decreasing order of stereo-specificity for the B isomer reflects primarily the decreasing ratios of nucleotide complexes coordinated to O rather than S on the beta-P as the metal ion is changed from Mg2+ to Co2+ to Cd2+. The kinetic parameters for the hexokinases have also been determined for adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) using the same three metal ions as activators. The A diastereomer is the preferred substrate regardless of the metal ion. This absence of reversal of stereospecificity for metal-ATP alpha S suggests that the alpha-P is not involved in coordinating the metal on the enzyme, unlike the beta-P. That is, the structural constraints of the ATP-binding site on the enzyme overcome the preferred coordination of Cd2+ to S. Given the greater stability of bidentate metal-ATP complexes over monodentate, these data are interpreted as indicating that MgATP binds to the mammalian hexokinases as the beta gamma-bidentate complex in the A screw sense geometry, as has been found for the yeast hexokinase (Jaffe and Cohn, reference cited above).

Animals↗
PubMedDate not supplied

Neonatal nutritional requirements and formula composition: a review.

Adequate neonatal nutrition is essential for appropriate growth and avoidance of complications associated with deficiencies. Neonatal nutritional requirements vary with metabolic states, degree of prematurity, and diseases affecting the gastrointestinal, pulmonary, and cardiac systems. Differences exist between formula brands for protein, fat, and carbohydrate sources. Differences also exist between formulas designed for preterm and full-term infants, as well as infants with special nutritional needs. Optimal nutrition should be a consistent goal for each infant, whether in a healthy newborn nursery, intensive-care unit, or home care setting. Nurses must be aware of the possibilities available to address the special needs of infants.

Dietary Carbohydrates↗