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A Sarai

Publications and source records attributed to A Sarai.

At least 73 records · Page 4Linked to original sources
PubMed1994

A simple method for assessing the mutational effect on the protein-DNA interaction: application to amino acid substitutions.

We have developed a simple method to assess the effect of mutations on the binding affinity change in the protein-DNA interaction. In this method, we evaluate the contribution of the local geometric effect in protein-DNA recognition by calculating the change in interaction energy caused by mutations, and assess the binding activity by introducing the binding affinity index. We applied this method to the interaction between lambda repressor and DNA. In spite of the simplicity of the present method, the calculated results qualitatively agree with experimental results for a limited number of mutants of lambda repressor. The mutational effects are mostly attributed to the complementary interactions in the protein-DNA complex. Although the method is still preliminary and should be the subject of further experimental verification, it may be used effectively at the planning stage of mutagenesis experiments to narrow down the selection of mutation by excluding unreasonable mutations.

Amino Acid Sequence↗
PubMed1993

Recognition of specific DNA sequences by the c-myb protooncogene product: role of three repeat units in the DNA-binding domain.

The DNA-binding domain of c-Myb consists of three homologous tandem repeats of 52 amino acids. The structure of the third (C-terminal) repeat obtained by NMR analysis has a conformation related to the helix-turn-helix motif. To identify the role of each repeat in the sequence recognition of DNA, we analyzed specific interactions between c-Myb and DNA by measuring binding affinities for systematic mutants of Myb-binding DNA sites and various truncated c-Myb mutants. We found that specific interactions are localized unevenly in the AACTGAC region in the consensus binding site of c-Myb: The first adenine, third cytosine, and fifth guanine are involved in very specific interactions, in which any base substitutions reduce the binding affinity by > 500-fold. On the other hand, the interaction at the second adenine is less specific, with the affinity reduction in the range of 6- to 15-fold. The seventh cytosine involves a rather peculiar interaction, in which only guanine substitution abolishes the specific binding. The binding analyses, together with the chemical protection analyses, showed that the c-Myb fragment containing the second and third repeats covers the AACTGAC region from the major groove of DNA in such an orientation that the third repeat covers the core AAC sequence. These results suggest that the third repeat recognizes the core AAC sequence very specifically, whereas the second repeat recognizes the GAC sequence in a more redundant manner. The first (N-terminal) repeat, which covers the major groove of DNA only partially, is not significant in the sequence recognition, but it contributes to increase the stability of the Myb-DNA complex. The presence of an N-terminal acidic region upstream of the first repeat, which is important for the activation of c-myb protooncogene, was found to reduce the binding affinity by interfering with the first repeat in binding to DNA.

Base Sequence↗
PubMed1993

Negative autoregulation of c-Myb activity by homodimer formation through the leucine zipper.

The trans-activating and transforming capacities of the c-myb proto-oncogene product (c-Myb) are negatively regulated through a leucine zipper structure in its negative regulatory domain. We show here tht in cotransfection assays, maximal Myb-induced trans-activation occurs with relatively low amounts of wild-type c-Myb, while higher levels of c-Myb result in reduced Myb-induced trans-activation. By contrast, this apparent negative autoregulation is not observed with a c-Myb mutant containing an impaired leucine zipper. Data presented here suggest that this negative autoregulation of trans-activation by wild-type c-Myb is a consequence of homodimer formation by c-Myb through its leucine zipper and of the inability of c-Myb dimers to bind DNA. These findings point to a novel mechanism of regulation of a transcription factor.

Amino Acid Sequence↗
PubMed1993

Thermodynamic and kinetic studies of DNA triplex formation of an oligohomopyrimidine and a matched duplex by filter binding assay.

The filter binding method was found to be a powerful method for studying the formation of triplexes composed of a single-stranded homopyrimidine and a duplex with a matched purine-pyrimidine tract. With this technique, we were able to determine thermodynamic and kinetic parameters for triplex formation between a homopyrimidine 19-mer (5'-TCCTCTTCTTTTCTTTCTT-3') and a duplex with sequence 5'-GCAGGAGAAGAAAAGAAAGAACG-3' for the purine strand. The experiments were performed over a wide pH range (3.8-7.4) and a temperature range of 0-35 degrees C. pH and temperature dependencies of the thermodynamic parameters were best explained in terms of a three-state model for triplex formation at low temperatures relative to the melting point. The main results were as follows: (1) pH dependence of the dissociation constants of the triplex is a result of the rapid acid-base equilibrium of pyrimidine single strands; (2) the association rate for triplex formation decreases with increasing pH in accordance with the dissociation constants; (3) the dissociation constant is virtually temperature-independent at low pH, while it becomes strongly temperature-dependent with increasing pH (these results can be explained in terms of a negative, non-zero delta Cp for triplex formation at low pH); (4) the association rate decreases with increasing temperature, and the resulting negative activation energy indicates that the triplex formation process involves a quasi-stable intermediate; (5) the triplex formation is a second-order reaction at low pH, whereas it can be interpreted as a third-order reaction at neutral pH, suggesting that different triplex formation pathways are observed depending on the pH.

Base Sequence↗
PubMed1993

Thermal stability of the DNA-binding domain of the Myb oncoprotein.

The DNA-binding domain of the c-myb protooncogene product consists of three homologous tandem repeats of 51-52 amino acids (denoted as R1, R2, and R3 from the N-terminal side). In order to analyze conformational and thermodynamic characteristics of the homologous repeats, we have examined the DNA-binding domain by circular dichroism (CD) and differential scanning calorimetry (DSC). The CD spectra for the three individual repeats are significantly different in the fine profiles, indicating subtle differences in their conformations. The melting analyses for the fragments show that the thermal stability of each fragment is different from one another, with the following order of stability: R1(Tm = 61 degrees C) approximately greater than R3(57 degrees C) >> R2(43 degrees C), where R2 is much less stable than the other repeats. The denaturing process for the whole DNA-binding domain, measured by DSC, is characterized by a very broad transition ranging from 30 to 80 degrees C. The denaturation curve can be fit well by a three-state transition with one intermediate state. The transition temperature for the native-to-intermediate transition coincides with the melting temperature of R2, indicating that the intermediate state corresponds to the unfolding of unstable R2. The CD spectrum of the whole domain is almost identical to the sum of the individual spectra. Thus, these results suggest that the individual repeats in the whole DNA-binding domain behave independently in terms of conformation and stability. The addition of DNA to the DNA-binding fragment drastically changed the melting profile, in which the broad transition curve was replaced by a sharp peak at 58 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Missense mutations in the Bacillus subtilis gnt repressor that diminish operator binding ability.

The Bacillus subtilis gnt operon is negatively regulated by the gnt repressor (GntR, 243 amino acids), which is antagonized by gluconate. The GntR protein belongs to a new family of bacterial regulatory proteins (GntR family). To locate the DNA-binding domain of the GntR protein, we obtained mutations of this protein, by hydroxylamine mutagenesis, which diminish its operator binding ability. Sequence analysis of these mutations indicated that the mutant GntR proteins (GntR43L, GntR66T, GntR74K and GntR75Q) had amino acid substitutions (Ser43 to Leu, Ala66 to Thr, Glu74 to Lys and Arg75 to Gln), respectively. They were all located within the N-terminal conserved region of the GntR family. In vivo and in vitro analysis of these GntR proteins indicated that their relative operator binding abilities became weaker in the order of GntR (wild type), GntR66T, GntR75Q, GntR74K and GntR43L. The equilibrium dissociation constants of GntR (wild type), GntR66T, GntR75Q and GntR74K as to operator binding were determined by gel retardation assays to be 0.43, 2.6, 4.2 and 8.8 M x 10(-10), respectively.

Amino Acid Sequence↗
PubMed1993

Different effects of substitutions at residues 224 and 228 of MHC class I on the recognition of CD8.

Previous studies indicated that weak xenoresponse to HLA class I by mouse T cells is due to the inefficient interaction of mouse CD8 with the alpha 3 domain of HLA class I. The present study using chimeric H-2Kb molecules with recombinant alpha 3 domain between H-2Kb and HLA-B7 as well as single amino acid mutants of H-2Kb demonstrated that each substitution at residues 224 and 228 affects recognition of CD8-dependent mouse CTL clones. On the other hand, reactivity of IL-2-producing H-2Kb-specific T cell hybridoma transfected with mouse CD8 alpha was abrogated by substitution at residue 224 but not by that at residue 228. This indicates that the substitution at residue 228 affects recognition of CD8-dependent CTL but does not critically affect binding of CD8 to MHC class I molecules, although residue 224 abrogates binding of CD8. The model structure of the alpha 3 domain of H-2Kb suggests that the substitution at residue 224 induces conformational change of CD8 binding loop, whereas minimum structure change by the substitution at residue 228 is expected. It is therefore speculated that minimum structure change of CD8 binding loop by substitution at residue 228 may influence binding affinity of CD8, which abrogates recognition of CD8-dependent CTL but not IL-2 production of the CD8-dependent T cell hybridoma.

Amino Acid Sequence↗
PubMed1993

Identification and differential expression of yeast SEC23-related gene (Msec23) in mouse tissues.

We have isolated a yeast SEC23-related clone (Msec23) from mouse fibroblast cDNA library. It has an open reading frame of 1721 bp (64% homologous to SEC23-2.3 kb) which can potentially encode a 64.7 kDa protein (61% homologous to 85.4 kDa product of SEC23). The deduced Msec23 protein (Msec23p) sequence contains three successive Ig-like domains at the N-terminus followed by amphipathic alpha-helical regions, suggesting the potential of Msec23p to interact with other protein components. Further, Msec23 is differentially expressed in mouse tissues with its high level in brain and fibroblasts.

Amino Acid Sequence↗
PubMed1993

A mathematically designed STS primer without any mismatches for direct sequencing of cosmid DNA clones.

We report here a new method for the direct sequencing of large DNA inserts of cosmid clones from human chromosomes using STS primers of 14 nucleotides without any mismatches, which are designed from results of a mathematical analysis. It is clear that STS primer of 14 nucleotides is optimum for direct sequencing of cosmid recombinant DNA clones. We also provide examples of direct sequencing of cosmid clones of human chromosome 21 using these STS primers.

Animals↗
PubMed1993

Thermodynamic and kinetic analyses of DNA triplex formation: application of filter-binding assay.

We have developed a simple and efficient method for studying equilibrium thermodynamics and kinetics of DNA triplex formation, which utilizes a filter-binding procedure. The application of this method to the triplex formation between a double-stranded homopurine-homopyrimidine and a single-stranded homopyrimidine oligonucleotides has demonstrated its ability in the quantitative estimation of equilibrium binding constants and rate constants under various conditions. Thus, this simple method can serve as a powerful tool for the systematic analysis of sequence and environmental effects on the equilibrium and kinetic quantities in the triplex formation.

Base Sequence↗
PubMed1993

Mechanism of DNA triplex formation and its specificity as studied by filter binding assay.

The filter binding method was found to be a very useful method for triple-helical formation of oligo DNA duplexes with homoprymidine single-strands. Using this method, we have obtained dissociation constants of triplexes and association rates of triple-helical formation of a variety of combinations of double-strands (23-mer) and pyrimidine single-strands as functions of pH and temperature. pH dependences of dissociation constants and association rates are theoretically discussed in terms of acid-base equilibrium of pyrimidine strands. Temperature-dependence of dissociation constants and association rates were considerably different between acidic and neutral pH range, suggesting that mechanism of triple-helical formation differs between two pH ranges. The results were best interpreted in terms of a three-state model for the triple-helical formation. Furthermore, the effect of mismatched sequences on the stability of the triplexes were also discussed.

Base Sequence↗
PubMed1993

Thermodynamics of tetraplex DNAs, (T2G4)n and (T4G4)n.

The ends of eukaryotic chromosomes, termed telomeres, contain stretches of tandemly repeated guanine-rich sequences, such as (T2G4)n and (T4G4)n, along one strand. These sequences can form defined folded tetraplex structures in solution. Here we have systematically investigated the thermodynamic properties of a series of sequences, Tet n: (T2G4)n and Oxy n: (T4G4)n (n = 1, 2, 3, 4), in 10mM NaPi, 250mM NaCl, pH7.0, using differential scanning calorimetry (DSC). The melting process of all tetraplex DNAs is reversible. Tet n and Oxy n are similar in the dependence of melting temperature, Tm, and transition calorimetric enthalpy per strand, delta Hcal, on the number of the tandem sequence. The total delta Hcal value of Tet n is similar in magnitude to that of Oxy n, whereas the Tm value of the main fraction of Tet n is greater than that of Oxy n. These results suggest that Tet n and Oxy n form similar tetraplex DNA configuration, and the difference in Tm values between Tet n and Oxy n is attributed to the length of the T strings.

Calorimetry, Differential Scanning↗
PubMed1993

Solution structures of Myb DNA-binding domain and its complex with DNA.

The DNA-binding domain of Myb consists of three imperfect tandem repeats, each of which contains three conserved tryptophans. The solution structure of the third repeat was established by NMR; it includes three helices maintained by a hydrophobic core formed by three tryptophans, together with two histidines, and the second and third helices form a helix-turn-helix-related motif. Here, we report the whole structure of the DNA-binding domain, as determined by NMR. Each of the first and second repeats resembles the third one in structure, retaining three helices. As a whole, the DNA-binding domain contains an entirely unique structure, a triple helix-turn-helix-related motif. To determine the role of each repeat in DNA-binding, a 16-mer DNA duplex that is specifically recognized by Myb was synthesized and then its complex with the DNA-binding domain has been investigated by 3D-NMR. The interaction mode between the whole DNA-binding domain and the duplex has shown that the third helix in each repeat likely locates in the majour groove of DNA. It is suggested that the Myb DNA-binding domain wraps around the DNA duplex by fitting the triply repeated helix-turn-helix-related motifs in the majour groove.

Base Sequence↗
PubMed1992

Solution structure of a DNA-binding unit of Myb: a helix-turn-helix-related motif with conserved tryptophans forming a hydrophobic core.

The DNA-binding domain of the c-myb protooncogene product consists of three imperfect tandem repeats of 51 or 52 amino acids, each of which contains three conserved tryptophans, spaced 18 or 19 amino acids apart. The structure of the third repeat, which is essential for sequence-specific DNA binding, has been determined by NMR with distance geometry calculation. It includes three well-defined helices (residues 149-162, 166-172, and 178-187) maintained by a hydrophobic core that includes the three conserved tryptophans, together with two histidines. Helices 2 and 3 form a structure related to but distinct from a canonical helix-turn-helix motif. In particular, the turn between these helices is one amino acid longer than the corresponding turn in bacterial repressors and homeodomains and contains a proline residue. In addition, the architecture of the three helices is different from those of homeodomains and DNA-binding domains of bacterial repressors. Based on the present structure, the binding mode of Myb repeat 3 with a specific DNA is also discussed.

Amino Acid Sequence↗
PubMed1992

Transactivation and transformation by Myb are negatively regulated by a leucine-zipper structure.

The negative regulatory domain of the c-myb protooncogene product (c-Myb) normally represses transcriptional activation by c-Myb. We show here that a leucine-zipper structure is a component of the negative regulatory domain, because its disruption markedly increases both the transactivating and transforming capacities of c-Myb. We also demonstrate that this leucine-zipper structure can interact with cellular proteins. Our results suggest that an inhibitor that suppresses transactivation binds to c-Myb through the leucine zipper and that c-Myb can be oncogenically activated by missense mutation.

3T3 Cells↗
PubMed1992

Coevolution of cytokine receptor families in the immune and nervous systems.

Close relationships between the nervous system and immune systems at molecular levels have now become evident. Receptors for CDF/LIF and CNTF, i.e., factors which play important roles in the nervous system, share a close structural similarity to those for IL-6, which is a molecule acting in the immune system. Receptors for these three factors belong to a subtype of cytokine receptor family (class IB cytokine receptor). We have constructed a higher subdomain structure of the receptor for CDF/LIF based on its known primary structures. The receptor contains immunoglobulin and fibronectin-like domains, in addition to common domains of the cytokine receptor, similar to those cell surface molecules of the neural immunoglobulin gene super family. These domains appear to have similar structures to the immunoglobulin. These lines of evidence suggest that the class IB cytokine receptor was formed as a result of those fusion of the genes for a more primitive cytokine receptor IA and for the neural immunoglobulin super gene family, and that, likewise, many molecules regulating neural development and those which act in the immune system have a common evolutionary origin.

Amino Acid Sequence↗
PubMed1992

Interaction of a protooncogene product, Myb with DNAs.

The DNA-binding domain of Myb consists of three imperfect tandem repeats and the third one which is essential for sequence-specific binding was established to have a helix-turn-helix-related motif. DNA sequences recognized by Myb have been reported to contain TAACPy sequence. Here we have examined the details of Myb-binding sequence. Using DNAs with a single mutation on the various sites of two specific DNAs and some fragments of the DNA-binding domain of Myb, we have found that (i) in a specific DNA which contains only one AAC sequence, each AAC nucleotide is found to be essential for the specific binding of Myb, while any other mutations cause no serious binding loss, (ii) in a specific DNA which contains two AAC sequences separately, one AAC is not so important in the binding, and (iii) for the specific binding with DNA, at least both repeats 2 and 3 of Myb are required. These findings suggest that repeat 3 containing a helix-turn-helix-related structure recognizes the core AAC sequence and repeat 2 supports this recognition by interactions with phosphate groups of DNA.

Base Sequence↗