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K Mizuuchi

Publications and source records attributed to K Mizuuchi.

At least 55 records · Page 3Linked to original sources
PubMed1988

Novel strand exchanges in V(D)J recombination.

We describe novel products of V(D)J recombination in which signal sequences become joined to coding elements, in contrast to the standard reaction whose products are junctions of two signal sequences or two coding elements. In this variant reaction, the recombination machinery evidently recognizes signal sequences and introduces strand breaks at the normal positions, but then connects the elements in unusual combinations. The lack of fixed directionality indicates that recombination sites are not uniquely aligned when strand exchange occurs. The discovery of these variant junctions suggests a model for the evolution of the antigen receptor loci.

Animals↗
PubMed1988

The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination.

Pre-B and pre-T cell lines from mutant mice with severe combined immune deficiency (scid mice) were transfected with plasmids that contained recombination signal sequences of antigen receptor gene elements (V, D, and J). Recovered plasmids were tested for possible recombination of signal sequences and/or the adjacent (coding) sequences. Signal ends were joined, but recombination was abnormal in that half of the recombinants had lost nucleotides from one or both signals. Coding ends were not joined at all in either deletional or inversional V(D)J recombination reactions. However, coding ends were able to participate in alternative reactions. The failure of coding joint formation in scid pre-B and pre-T cells appears sufficient to explain the absence of immunoglobulin or T cell receptor production in scid mice.

Animals↗
PubMed1988

Retroviral DNA integration: structure of an integration intermediate.

The structure of a presumptive DNA intermediate in the integration of retroviral DNA was studied in a cell-free reaction with exogenously added target DNA. The product made by viral core particles of Moloney murine leukemia virus (Mo-MLV) containing linear viral DNA has a structure consistent with an integration mechanism similar to that observed during bacteriophage Mu transposition. In this intermediate, the 3' ends of the LTR sequences are joined to the target DNA, while the 5' ends of the viral DNA remain unjoined. The 5' ends of the LTR sequences in the intermediate are exactly the same as those found in the unintegrated linear double-stranded viral DNA. This result demonstrates that the linear form of Mo-MLV DNA can integrate directly without prior circularization.

Cell-Free System↗
PubMed1988

Target immunity of Mu transposition reflects a differential distribution of Mu B protein.

A DNA molecule carrying Mu end DNA sequence(s) is a poor target in the Mu DNA strand-transfer reaction, a phenomenon which is referred to as "target immunity." We find that Mu B protein stimulates intermolecular strand-transfer by binding to the target DNA. Our results show that a differential distribution of Mu B protein between "immune" and "non-immune" DNA molecules is responsible for target immunity; in the presence of Mu A protein and ATP, Mu B protein dissociates preferentially from immune DNA molecules. Hydrolysis of ATP is implicated in establishing the differential distribution of Mu B protein between immune and non-immune DNA molecules in the presence of Mu A protein; nonhydrolyzable ATP gamma S can support an efficient strand-transfer reaction even with a target DNA that is immune in a reaction with ATP.

Adenosine Triphosphatases↗
PubMed1988

Lymphoid V(D)J recombination: nucleotide insertion at signal joints as well as coding joints.

The coding regions of antigen receptor genes assembled by variable-diversity-joining region [V(D)J] recombination are known in many cases to have undergone deletions of several nucleotides and also to contain insertions of noncoded nucleotides at the recombined junction (the coding joint). By using extrachromosomal recombination substrates to transfect lymphoid cell lines, we show that the signal joint (the fusion of the corresponding recognition signal sequences) can also contain insertions; however, nucleotide loss from the signals is very rare. The frequency of nucleotide addition varies among pre-B-cell lines in a manner proportional to their content of terminal deoxynucleotidyltransferase. We also find frequent nucleotide additions (and deletions) at coding joints, but in this case there is no strong correlation with the level of terminal deoxynucleotidyltransferase activity. Inserts at both signal and coding joints are rich in G + C, consistent with the base utilization preference of this enzyme.

Animals↗
PubMed1987

Transposition of Mu DNA: joining of Mu to target DNA can be uncoupled from cleavage at the ends of Mu.

Transposition of Mu involves transfer of the 3' ends of Mu DNA to the 5' ends of a staggered cut in the target DNA. We find that cleavage at the 3' ends of Mu DNA precedes cutting of the target DNA. The resulting nicked species exists as a noncovalent nucleoprotein complex in which the two Mu ends are held together. This cleaved donor complex completes strand transfer when a target DNA, Mu B protein, and ATP are provided. Mu end DNA sequences that have been precisely cut at their 3' ends by a restriction endonuclease, instead of by Mu A protein and HU, are efficiently transferred to a target DNA upon subsequent incubation with Mu A protein, Mu B protein, and ATP. Cleavage of the Mu ends therefore cannot be energetically coupled with joining these ends to a target DNA. We discuss the DNA strand transfer mechanism in view of these results, and propose a model involving direct transfer of the 5' ends of the cut target DNA, from their original partners, to the 3' ends of Mu.

Coliphages↗
PubMed1987

Extrachromosomal DNA substrates in pre-B cells undergo inversion or deletion at immunoglobulin V-(D)-J joining signals.

Sequences encoding immunoglobulin variable domains are known to be assembled from variable (V), diversity (D), and joining (J) segments by site-specific recombination. We present a sensitive and rapid assay for V-(D)-J recombination that uses plasmid DNA transiently introduced into transformed pre-B cells, and demonstrates that the recombination is independent of any unique chromosomal context. Sequences sufficient to constitute recombination sites are contained within the 84 and 42 bp flanking, respectively, the murine J kappa 1 and V kappa L8 segments, which include the known heptamer-nonamer V-(D)-J joining signals. Deletion and inversion occur at comparable frequencies. Thus, V-(D)-J recombination may be relatively insensitive to the topological arrangement of sites, and events at the two novel junctions produced by the reaction may be coupled.

Animals↗
PubMed1987

DNA sequence of the E. coli gyrB gene: application of a new sequencing strategy.

We have determined the sequence of the E. coli gyrB gene, using a new sequencing approach in which transposition from a mini-Mu plasmid into the DNA provides random start points for dideoxynucleotide sequence analysis. The gyrB sequence corresponds to a protein 804 amino acids long; a previously isolated protein fragment with partial enzymatic activity has been identified as the C-terminal half-molecule. A plausible terminator of gyrB transcription is located just beyond the structural gene.

Amino Acid Sequence↗
PubMed1987

B protein of bacteriophage mu is an ATPase that preferentially stimulates intermolecular DNA strand transfer.

A DNA strand-transfer reaction is an early step in the transposition of phage Mu. It has been shown that an efficient reaction in vitro requires, in addition to buffer and salt, only the Mu A protein, Mu B protein, host protein HU, ATP, and Mg2+. We have determined that, of the three protein factors involved, only the Mu B protein has an ATPase activity. The Mu B ATPase is stimulated by Mu A protein and DNA but not by either of these factors alone. Double-stranded DNA is a much better cofactor than single-stranded DNA, but there is no apparent sequence specificity. In the absence of the Mu B protein and/or ATP, the intermolecular Mu DNA strand-transfer reaction is extremely inefficient, and the strand-transfer products are predominantly the result of an intramolecular reaction. This contrasts with the efficient intermolecular reaction that occurs if Mu B protein and ATP are provided. The Mu B protein, in the presence of Mu A protein and protein HU, therefore, seems to facilitate interactions between potential DNA target sites and pairs of Mu DNA ends.

Adenosine Triphosphatases↗
PubMed1987

Developmental stage specificity of the lymphoid V(D)J recombination activity.

We have examined the level of immunoglobulin gene V(D)J recombination activity in a number of cell lines derived from lymphoid or nonlymphoid lineages. The assay we employed uses extrachromosomal DNA as substrate and thereby avoids difficulties associated with the use of chromosomally integrated substrates. The recombination activity decreases during B-lymphoid development. It is highest at the earliest stages of committed B-cell differentiation and then falls progressively, reaching undetectable levels at the mature B-cell stage. The activity is also present in multipotential progenitors of myeloid cells and in pre-T cells but not mature T cells. No activity was found in several nonhematopoietic cell lines. Recombination was seen only among substrate molecules which had replicated in the eukaryotic cells. Several possible interpretations of this result are discussed.

Animals↗
PubMed1986

Role of DNA topology in Mu transposition: mechanism of sensing the relative orientation of two DNA segments.

DNA strand transfer at the initiation of Mu transposition normally requires a negatively supercoiled transposon donor molecule, containing both ends of Mu in inverted repeat orientation. We propose that the specific relative orientation of the Mu ends is needed only to energetically favor a particular configuration that the ends must adopt in a synaptic complex. The model was tested by constructing special donor DNA substrates that, because of their catenation or knotting, energetically favor this same configuration of the Mu ends, even though they are on separate molecules or in direct repeat orientation. These structures are efficient substrates for the strand transfer reaction, whereas appropriate control structures are not. The result eliminates tracking or protein scaffold models for orientation preference. Several other systems in which the relative orientation of two DNA segments is sensed may utilize the same mechanism.

Bacteriophage mu↗
PubMed1986

DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu.

We have determined the DNA sequence of the control region of phage D108 up to position 1419 at the left end of the phage genome. Open reading frames for the repressor gene, ner gene, and the 5' part of the A gene (which codes for transposase) are found in the sequence. The genetic organization of this region of phage D108 is quite similar to that of phage Mu in spite of considerable divergence, both in the nucleotide sequence and in the amino acid sequences of the regulatory proteins of the two phages. The N-terminal amino acid sequences of the transposases of the two phages also share only limited homology. On the other hand, a significant amino acid sequence homology was found within each phage between the N-terminal parts of the repressor and transposase. We propose that the N-terminal domains of the repressor and transposase of each phage interact functionally in the process of making the decision between the lytic and the lysogenic mode of growth.

Amino Acid Sequence↗
PubMed1986

Crystallization of a DNA tridecamer d(C-G-C-A-G-A-A-T-T-C-G-C-G).

Crystals of the DNA tridecamer d(C-G-C-A-G-A-A-T-T-C-G-C-G) have been grown by the vapor-diffusion technique with 2-methyl-2,4-pentanediol as precipitant. They are monoclinic space group C2, with a = 79.6 A, b = 43.1 A, c = 24.9 A and beta = 98.7 degrees. Previous nuclear magnetic resonance studies predicted that this tridecamer forms a duplex similar to the B DNA dodecamer, d(C-G-C-G-A-A-T-T-C-G-C-G), except for an extra adenosine residue that is stacked within the helix but remains unpaired: (formula; see text) Preliminary X-ray diffraction studies confirmed that the tridecamer is in the B DNA conformation, consistent with the nuclear magnetic resonance results.

Crystallography↗
PubMed1985

The extent of DNA sequence required for a functional bacterial attachment site of phage lambda.

We have investigated the extent of DNA sequence required to form a bacterial attachment site (attB) that functions in bacteriophage lambda integration. A DNA fragment carrying attB of Escherichia coli was trimmed, recloned and tested for recombination proficiency. We found that the common core sequence plus the adjoining 4-bp sequences of both the B and B' arms are required for full activity, while plasmids with an even shorter attB sequence retain some capacity to function as attB in vivo. We also found that the nonspecific DNA that is joined to the required attachment site sequence does not significantly influence the rate of the recombination reaction.

Bacteriophage lambda↗
PubMed1985

Cloning of the A gene of bacteriophage Mu and purification of its product, the Mu transposase.

The bacteriophage Mu transposase (the Mu A gene product), which is absolutely required for both integration of Mu and replicative transposition during the lytic cycle, has been overproduced by cloning the gene on a plasmid under the control of the phage lambda PL promoter. The protein has been purified to near homogeneity from the lysate of heat-induced cells of a strain carrying the plasmid. The purified protein is active as judged by its ability to complement Mu A- cell extracts for supporting Mu transposition in a cell-free reaction.

Bacteriophage lambda↗
PubMed1985

Mechanism of transposition of bacteriophage Mu: structure of a transposition intermediate.

Mu transposition works efficiently in vitro and generates both cointegrate and simple insert products. We have examined the reaction products obtained under modified in vitro reaction conditions that do not permit efficient initiation of DNA replication. The major product is precisely the intermediate structure predicted from one of the current models of DNA transposition. Both cointegrates and simple inserts can be made in vitro using this intermediate as the DNA substrate, demonstrating that it is indeed a true transposition intermediate. The requirements for efficient formation of the intermediate include the Mu A protein, the Mu B protein, an unknown number of E. coli host proteins, ATP, and divalent cation. Only E. coli host proteins are required for conversion of the intermediate to cointegrate or simple insert products. Structures resulting from DNA strand transfer at only one end of the transposon are not observed, suggesting that the strand transfers at each end of the transposon are tightly coupled.

Adenosine Triphosphate↗