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Biomedical subjects

M Gellert

Publications and source records attributed to M Gellert.

At least 73 records · Page 4Linked to original sources
PubMed1992

V(D)J recombination in mouse thymocytes: double-strand breaks near T cell receptor delta rearrangement signals.

In the murine T cell receptor delta locus, V(D)J recombination events frequently involve the D2 and J1 elements. Here we report the presence of double-strand breaks at recombination signals flanking D2 in approximately 2% of thymus DNA. An excised linear species containing the sequences between D2 and J1 and a circular product of the joining of D2 and J1 recombination signals were also found. Although broken molecules with signal ends were detected, no species with coding ends could be identified. Observation of these broken molecules in thymus, but not in liver or spleen, provides the first direct evidence for an association between specific cleavage of chromosomal DNA and recombination in mammalian cells, and supports a breakage-reunion model of V(D)J recombination.

Animals↗
PubMed1992

V(D)J recombination gets a break.

The diversity of immunoglobulins and T cell receptors is largely due to the assembly of functional genes from separate segments. The mechanism by which these gene fragments are joined is starting to be deciphered, with broken DNA molecules that may be intermediates in the reaction providing a new clue.

Animals↗
PubMed1990

Characterization of the ATP binding site on Escherichia coli DNA gyrase. Affinity labeling of Lys-103 and Lys-110 of the B subunit by pyridoxal 5'-diphospho-5'-adenosine.

We have labeled the adenosine triphosphate binding site of Escherichia coli DNA gyrase with the ATP affinity analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP strongly inhibits the ATP-ase and DNA supercoiling activities of DNA gyrase, with 50% inhibition occurring at 7.5 microM inhibitor. ATP and ADP compete with PLP-AMP for binding and protect the enzyme against inhibition. The labeling appears to proceed by a Schiff base complex between the 4-formyl group of the pyridoxyl moiety of PLP-AMP and a protein primary amino group, since the inhibition and reagent labeling are reversible unless the complex is treated with NaBH4. Complete inactivation is estimated to occur upon the covalent incorporation of 2 mol of inhibitor/mol of gyrase. The Km for ATP was found to be unchanged for partially inhibited enzyme samples, suggesting an all-or-none type of inhibition. A 3H-labeled peptide spanning residues 93-131 of the B protein was isolated from a V-8 protease digest. Radioactive peaks corresponding to Lys-103 and Lys-110 were found during the Edman degradation, suggesting that these amino acids form part of the ATP binding site. A comparison of the amino acid sequence in this region with the sequences of other type II topoisomerases indicates the possible location of a common ATP binding domain.

Adenosine Diphosphate↗
PubMed1990

Neutron and light-scattering studies of DNA gyrase and its complex with DNA.

The solution structure of Escherichia coli DNA gyrase, an enzyme that catalyzes the ATP-dependent supercoiling of DNA, has been characterized by small-angle neutron scattering (SANS) and dynamic light-scattering (DLS). The enzyme and its complex with a 172 base-pair fragment of duplex DNA, in H2O or 2H2O solvent, were studied by contrast variation and the measurement of hydrodynamic parameters as a function of scattering angle. The complex was also measured in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP), a non-hydrolyzable ATP analog that is known to support limited supercoiling. The values of the radius of gyration, Rg = 67 A, from SANS and the hydrodynamic radius, Rh = 64 A, from DLS predict a larger than expected volume for the enzyme, supporting the notion of channels or cavities within the molecule. In addition, several classes of models were rejected based on SANS data obtained in 2H2O at larger scattering angles. The best fit to both the SANS and DLS data is obtained for oblate, inhomogeneous particles approximately 175 A wide and 52 A thick. Such particles provide a large surface area for DNA interaction. Both Rg and Rh values change very little upon addition of DNA, suggesting that DNA binds in a manner that does not significantly change the shape of the protein. No appreciable change in structure is found with the addition of ADPNP. However, the higher-angle SANS data indicate a slight rearrangement of the enzyme in the presence of nucleotide.

Animals↗
PubMed1990

V(D)J recombination activity in lymphoid cell lines is increased by agents that elevate cAMP.

V(D)J [variable--(diversity)--joining] recombination is regulated developmentally, being restricted to cells of the early B- and T-lymphocyte lineages. In this report we show that recombination activity can also be regulated in response to chemical effectors. Compounds that increase intracellular cAMP increase V(D)J recombination of extrachromosomal substrates in pre-B-cell lines as much as 10-fold. In contrast, V(D)J recombination is reduced 5- to 8-fold in response to phorbol 12-myristate 13-acetate or to the calcium ionophore A23187. The effect of cAMP agonists on recombination appears to reflect an increase in cellular recombination activity, as indicated by the caffeine-induced rise in the level of mRNA from the recombination-activating genes RAG1 and RAG2. Our data demonstrate that intracellular second messengers modulate recombination activity in lymphoid cell lines, implying that recombination activity can be regulated by these signals in developing B and T lymphocytes.

1-Methyl-3-isobutylxanthine↗
PubMed1989

V(D)J recombination: a functional definition of the joining signals.

Two conserved DNA sequences serve as joining signals in the assembly of immunoglobulins and T-cell receptors from V-, (D)-, and J-coding segments during lymphoid differentiation. We have examined V(D)J recombination as a function of joining signal sequence. Plasmid substrates with mutations in one or both of the heptamer-spacer-nonamer sequences were tested for recombination in a pre-B-cell line active in V(D)J recombination. No signal variant recombines more efficiently than the consensus forms of the joining signals. We find the heptamer sequence to be the most important; specifically, the three bases closest to the recombination crossover site are critical. The nonamer is not as rigidly defined, and it is not important to maintain the five consecutive As that distinguish the consensus nonamer sequence. Both types of signals display very similar sequence requirements and have in common an intolerance for changes in spacer length greater than 1 bp. Although the two signal types share sequence motifs, we find no evidence of a role in recombination for homology between the signals, suggesting that they serve primarily as protein recognition and binding sites.

Base Sequence↗
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

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

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

Structure of the DNA gyrase-DNA complex as revealed by transient electric dichroism.

We have analyzed the structure of complexes between DNA gyrase and four defined DNA fragments by electric dichroism. Both the extrapolated dichroism and relaxation time of these complexes suggest that a single turn of DNA is wrapped around the enzyme with the entry and exit points located close together. The average angle between the DNA tails emerging from the particle is about 120 degrees. This structure is consistent with that seen by electron microscopy. Addition of ATP or the non-hydrolyzable ATP analog 5'-adenylyl-beta, gamma-imidodiphosphate results in a structural change of the complex, consistent with the DNA tails now being wrapped around the protein. The significance of these observations with respect to the mechanism of DNA supercoiling by DNA gyrase is discussed.

Autoradiography↗
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

Modulation of transcription by DNA supercoiling: a deletion analysis of the Escherichia coli gyrA and gyrB promoters.

Expression of the genes determining the subunits of Escherichia coli DNA gyrase (gyrA and gyrB) is known to be induced by relaxation of the template DNA. In this paper we report a deletion analysis of the gyrA and gyrB promoter regions. We find that a DNA sequence 20 base pairs long that includes the -10 consensus region, the transcription start point, and the first few transcribed bases is responsible for the property of induction by DNA relaxation. We propose a model for relaxation-stimulated transcription in which promoter clearance is the rate-limiting step.

Base Sequence↗