A pivotal role for the structure of the Holliday junction in DNA branch migration.

Branch migration of a DNA Holliday junction is a key step in genetic recombination that affects the extent of transfer of genetic information between homologous DNA sequences. We previously observed that the rate of spontaneous branch migration is exceedingly sensitive to metal ions and postulated that the structure of the cross-over point might be one critical determinant of the rate of branch migration. Other investigators have shown that in the presence of divalent metal ions like magnesium, the Holliday junction assumes a folded conformation in which base stacking is retained through the cross-over point. This base stacking is disrupted in the absence of magnesium. Here we measure the rate of branch migration as a function of Mg2+ concentration. The rate of branch migration increases dramatically at MgCl2 concentrations below 500 microM, with the steepest acceleration occurring between 300 and 100 microM MgCl2. This increase in the rate of branch migration coincides with the loss of base stacking in the four-way junction over this same interval of magnesium concentration, as measured by the susceptibility of junction residues to modification by osmium tetroxide and diethyl pyrocarbonate. We conclude that at physiological concentrations of intracellular Mg2+, base stacking in the Holliday junction constitutes one kinetic barrier to branch migration and that disruption of base stacking at the cross-over relieves this constraint.     

Determination of pilocarpic acid in human plasma by capillary gas chromatography with mass-selective detection

A novel, highly sensitive method for the determination of pilocarpic acid (PA) in human plasma is described. In addition, the method provides for the conversion of the lactone, pilocarpine (P), to PA so that a total drug presence can be determined. Using novel high-performance liquid chromatographic conditions capable of separating P, isopilocarpine (I-P), PA and isopilocarpic acid (I-PA) from each other and from endogenous plasma impurities, it was confirmed that P exclusively and quantitatively converts to PA in heparinized human plasma during storage. For the determination of PA, the selective extraction of PA from protein-free plasma was accomplished using two different solid-phase extraction (SPE) cartridges in two consecutive SPE steps. After extraction, PA was lactonized with trifluoroacetic acid back to P, and both P and an internal standard were acylated using heptafluorobutyric anhydride (HFBA). The trifluoroacetylated derivatives were monitored using gas chromatography (GC) with mass spectrometric (MS) detection. This procedure allowed the sensitive and reliable determination of PA with a limit of quantification (LOQ) of 1 ng/ml, which could not be achieved using previously described methods. The assay was validated in the concentration range of 1 to 10 ng/ml with an intra-day precision (expressed as the coefficient of variation, C.V.) ranging from 9.9 to 0.5%. Inter-day precision for the quality control standard at 2.5 ng/ml showed a C.V. of 10.2%. Accuracy ranged from 94 to 102%. The assay was used to monitor the maximum systemic exposure to P, administered by the ocular route, in terms of total plasma PA (P and PA).     

The human kininogen gene (KNG) mapped to chromosome 3q26-qter by analysis of somatic cell hybrids using the polymerase chain reaction

Summary Kinins, peptide products of kininogens, may be involved in hypertensive and diabetic diseases, and inflammatory disorders. The human kininogen gene (KNG) has been mapped to chromosome 3, using a panel of human-hamster somatic cell hybrids by polymerase chain reaction of hybrid DNA with gene-specific primers. KNG was further assigned to 3q26-3qter, using DNA from a second panel of chromosome 3 deletion mapping cell hybrids.     

Construction of expanded continuous life tables--a generalization of abridged and complete life tables.

This article extends the recent abridged life-table method of Hsieh. It generalizes the conventional discrete (abridged and complete) life tables into a continuous life table that can produce life-table functions at any age and develops a unified method of life-table construction that simplifies the disparate laborious procedures used in the traditional approach of constructing abridged and complete life tables. A set of precise procedures based on the complete cubic spline for the main body of the table and a mortality law for advanced ages is developed for estimating the basic and nonbasic life-table functions from a given mortality schedule. The proposed method can also produce more life-table functions than other existing methods. The method is illustrated with Canadian data.     

The Drosophila glutathione S-transferase 1-1 is encoded by an intronless gene at 87B

The Drosophila glutathione S-transferase 1-1 is a dimer of a 209 amino acid subunit, designated DmGST1. DmGST1 is encoded by a member of a multigene family. Sequence analysis of a genomic clone for GST1 revealed that it is encoded by an intronless gene. We designate this gene and its other family members the GST D genes in the glutathione S-transferase gene superfamily. The Drosophila GST D genes are mapped by in situ hybridization to chromosome 3R at 87B of the polytene chromosome, which is flanked by the two clusters of hsp70 genes at 87A7 and 87C1. Cytogenetic data in the literature indicated that a puff occurred in this region under heat shock. We report that the glutathione S-transferase activity in Kco cells as determined by conjugation with 1-chloro-2,4-dinitrobenzene is elevated slightly to two-fold under heat shock. The implication of this finding is discussed.     

Nuclease protection by Drosophila DNA topoisomerase II. Enzyme/DNA contacts at the strong topoisomerase II cleavage sites

A DNA consensus sequence for topoisomerase II cleavage sites was derived previously based on a statistical analysis of the nucleotide sequences around 16 sites that can be efficiently cleaved by Drosophila topoisomerase II (Sander, M., and Hsieh, T. (1985) Nucleic Acids Res. 13, 1057-1072). A synthetic 21-mer DNA sequence containing this cleavage consensus sequence was cloned into a plasmid vector, and DNA topoisomerase II can cleave this sequence at the position predicted by the cleavage consensus sequence. DNase I footprint analysis showed that topoisomerase II can protect a region of approximately 25 nucleotides in both strands of the duplex DNA, with the cleavage site located near the center of the protected region. Similar correlation between the DNase I footprints and strong topoisomerase II cleavage sites has been observed in the intergenic region of the divergent HSP70 genes. This analysis therefore suggests that the strong DNA cleavage sites of Drosophila topoisomerase II likely correspond to specific DNA-binding sites of this enzyme. Furthermore, the extent of DNA contacts made by this enzyme suggests that eucaryotic topoisomerase II, in contrast to bacterial DNA bacterial DNA gyrase, cannot form a complex with extensive DNA wrapping around the enzyme. The absence of DNA wrapping is probably the mechanistic basis for the lack of DNA supercoiling action for eucaryotic topoisomerase II.     

CpG methylated minichromosomes become inaccessible for V(D)J recombination after undergoing replication.

The physical parameters controlling the accessibility of antigen receptor loci to the V(D)J recombination activity are unknown. We have used minichromosome substrates to study the role that CpG methylation might play in controlling V(D)J recombination site accessibility. We find that CpG methylation decreases the V(D)J recombination of these substrates more than 100-fold. The decrease correlates with a considerable increase in resistance to endonuclease digestion of the methylated minichromosome DNA. The minichromosomes acquire resistance to both the intracellular V(D)J recombinase and exogenous endonuclease only after DNA replication. Therefore, CpG methylation specifies a chromatin structure that, upon DNA replication, is resistant to eukaryotic site-specific recombination. These findings are important to V(D)J recombination as well as to the chromatin assembly of methylated DNA during replication.     

Analysis of the defect in DNA end joining in the murine scid mutation.

Murine severe combined immune deficiency (scid) is marked by a 5,000-fold reduction in coding joint formation in V(D)J recombination of antigen receptors. Others have demonstrated a sensitivity to double-strand breaks generated by ionizing radiation and bleomycin. We were interested in establishing the extent of the defect in intramolecular and intermolecular DNA end joining in lymphoid and nonlymphoid cells from scid mice. We conducted a series of studies probing the ability of these cells to resolve free ends of linear DNA molecules having various biochemical end configurations. We find that the stable integration of linear DNA into scid fibroblasts is reduced 11- to 75-fold compared with that in normal fibroblasts. In contrast, intramolecular and intermolecular end joining occur at normal frequencies in scid lymphocytes and fibroblasts. This normal level of end joining is observed regardless of the type of overhang and regardless of the requirement for nucleolytic activities prior to ligation. The fact that free ends having a wide variety of end configurations are recircularized normally in scid cells rules out certain models for the defect in scid. We discuss the types of DNA end joining reactions that are and are not affected in this double-strand break repair defect in the context of a hairpin model for V(D)J recombination.