Left-handed DNA is shown to exist and elicit a biological response in Escherichia coli. A plasmid encoding the gene for a temperature-sensitive Eco RI methylase (MEco RI) was cotransformed with different plasmids containing inserts that had varying capacities to form left-handed helices or cruciforms with a target Eco RI site in the center or at the ends of the inserts. Inhibition of methylation in vivo was found for the stable inserts with the longest left-handed (presumably Z) helices. In vitro methylation with the purified MEco RI agreed with the results in vivo. Supercoil-induced changes in the structure of the primary helix in vitro provided confirmation that left-handed helices were responsible for this behavior. The presence in vivo of left-handed inserts elicits specific deletions and plasmid incompatibilities in certain instances.

Our results imply that a BP susceptibility gene exists near the centromere of chromosome 18. Confirmation of this finding (by independent investigators studying different pedigrees) has been published, suggesting that a valid BP disease linkage may have been discovered.

The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.

A sequence-specific genomic delivery system for the correction of chromosomal mutations was designed by incorporating two different binding domains into a single-stranded oligonucleotide. A repair domain (RD) contained the native sequence of the target region. A third strand-forming domain (TFD) was designed to form a triplex by Hoogsteen interactions. The design was based upon the premise that the RD will rapidly form a heteroduplex that is anchored synergistically by the TFD. Deoxyoligonucleotides were designed to form triplexes in the human adenosine deaminase (ADA) and p53 genes adjacent to known point mutations. Transfection of ADA-deficient human lymphocytes corrected the mutant sequence in 1-2% of cells. Neither the RD or TFD individually corrected the mutation. Transfection of p53 mutant human glioblastoma cells corrected the mutation and induced apoptosis in 7.5% of cells.

No abstract

No abstract

No abstract

The cysteine proteinase cathepsin B is one member of the lysosomal acid hydrolases. Based on the peptide sequence of rat liver cathepsin B, an oligonudeotide mixture containing 128 different 17-mers was synthesized and used as a probe to screen adult and fetal human liver cDNA libraries. A recombinant clone with a 1540-nucleotide insert was identified from the fetal library, and DNA sequence analysis confirmed that this done encodes human cathepsin B. The clone, designated pCB-1, has sequences for 81% of the coding region (for amino acid residues 50-252) together with w880 nucleotides of the 3' untranslated region of the mRNA. The DNA sequence also shows that the predicted carboxyl terminus of the coding sequence is longer than the mature protein by 6 amino acid residues. Southern blot analysis of restriction enzyme digests of human placental DNA revealed a simple pattern of hybridizing fragments using the cathepsin B coding sequence as probe. The result suggests that there is a single copy of cathepsin B gene per haploid genome.Proteinases are present in all forms of living organisms. The events of general and limited proteolyses have been attributed to actions of these enzymes. In general proteolysis, proteinases digest nutrient proteins and participate in the turnover of cellular proteins, whereas in limited proteolysis, proteinases modify substrate proteins and alter the properties and physiological functions of these proteins. Thus, limited proteolysis can play regulatory roles during cell growth and differentiation (1,2).Based on the amino acid residues at their active sites, proteinases are classified into serine, cysteine, aspartate, and metalloproteinases. Members of the cysteine proteinase family have wide phylogenetic distribution, and examples include papain from the papaya plant and cathepsin B from mammalian tissues. Cathepsin B is a lysosomal enzyme that is functional during intracellular protein catabolism and may also be involved in the proteolytic processing of protein precursors such as proinsulin (3)(4)(5). Cathepsin B has been implicated in several disease states including muscular dystrophy (6), rheumatoid arthritis (7), and tumor metastasis (8-11). We and others have demonstrated that cathepsin B-like proteinases may also be involved during differentiation of the cellular slime mold Dictyostelium discoideum (12, 13).To further investigate the role of cathepsin B in cellular functions, we have selected the molecular genetic approach. Here we report our results on the isolation and characterization of a cDNA clone for human cathepsin B.MATERIALS AND METHODS Synthesis of Oligodeoxyribonucleotides. A mixture of 17-nucleotide-long oligonucleotides encoding a portion of the rat liver cathepsin B protein sequence was synthesized by using a solid-phase phosphotriester method (14, 15). For amino acids specified by more than one codon, all possible nucleotides were inserted at the ambiguous positions. The synthesis was performed with a Vega Model 280 Automated Polynucleotide Synthesizer by a...