A generic algorithm for finding restriction sites within DNA sequences

This paper describes a generic algorithm for finding restrictionsites within DNA sequences. The ‘genericity’ ofthe algorithm is made possible through the use of set theory.Basic elements of DNA sequences, i.e. nucleotides (bases), arerepresented in sets, and DNA sequences, whether specific, ambiguousor even protein-coding, are represented as sequences of thosesets. The set intersection operation demonstrates its abilityto perform pattern-matching correctly on various DNA sequences.The performance analysis showed that the degree of complexityof the pattern matching is reduced from exponential to linear.An example is given to show the actual and potential restrictionsites, derived by the generic algorithm, in the DNA sequencetemplate coding for a synthetic calmodulin. Received on October 2, 1990; accepted on December 18, 1990     

An isothermal titration calorimetric method to determine the kinetic parameters of enzyme catalytic reaction by employing the product inhibition as probe.

An isothermal titration calorimetric (ITC) method was developed to measure the kinetic parameters of ribonuclease A catalytic hydrolysis of cytidine 2',3'-cyclic monophosphate. Employing the inhibition of product as a probe, the K(m), K(i), k(c), and DeltaH(m) can be determined by two simple calorimetric measurements. First, the substrate was titrated into the cell containing high concentration of enzyme. The molar reaction heat was calculated from the titration peak area divided by substrate moles per titration, and the initial catalytic reaction rate in the presence of various concentrations of product can be calculated from the peak height and the molar reaction heat. From Michaelis-Menten function in the presence of inhibitors, the relationship between K(m) and K(i) can be obtained. Then, the dissociation constant, which is equal to K(i), was measured by a regular ITC experiment. Thus, K(m) and k(c) can be calculated. The method developed here can be applied in other enzyme catalytic systems with inhibitive products.     

Activation of the human epsilon- and beta-globin promoters by SV40 T antigen.

We have studied the effect of the SV40 T antigen on expression from human globin promoters fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and compared its effect with the SV40 enhancer and the adenovirus E1A protein. We have observed that expression of p epsilon GLCAT and p beta GLCAT (the epsilon-globin or beta-globin promoter linked to the CAT gene) was significantly stimulated when cotransfected with a cloned T antigen plasmid into CV-1 cells, indicating that trans-activation of the globin promoters was mediated by SV40 T antigen. Transfection of the p beta GLCAT-SV (p beta GLCAT containing the SV40 enhancer element) into CV-1 cells resulted in a 50-60-fold increase in CAT activity as compared to p beta GLCAT (no enhancer). However, cotransfection of the p beta GLCAT-SV with the cloned T antigen resulted in an additional increase of CAT expression, which suggests that T antigen and the SV40 enhancer activate globin gene expression independently. We found that T antigen but not E1A could further stimulate the expression of an enhancer-containing plasmid in CV-1 cells; whereas E1A but not T antigen could further stimulate p epsilon GLCAT expression in COS-1 cells which constitutively express the SV40 T antigen. These results suggest that T antigen and E1A also act independently. Deletion analysis showed that the minimum sequence required for a detectable level of stimulation of the epsilon-globin promoter by T antigen is 177 bp 5' to the cap site, suggesting that the target sequences for response to T antigen do not reside in the canonical 100 bp promoter region, but rather reside in sequences further upstream, and therefore the cellular factors interacting with T antigen are not the TATA or CAT box binding proteins, but the proteins interacting with upstream regulatory sequences.     

Classical Raman spectroscopic studies of NADH and NAD+ bound to lactate dehydrogenase by difference techniques

The binding of the coenzymes NAD+ and NADH to lactate dehydrogenase causes significant changes in the Raman spectra of both of these molecules relative to spectra obtained in the absence of enzyme. The molecular motions of the bound adenine moiety of both NAD+ and NADH as well as adenine containing analogues of these coenzymes produce Raman bands that are essentially identical, suggesting that the binding of adenine to the enzyme is the same regardless of the nicotinamide head-group nature. We also have observed that the molecular motions of the bound adenine moiety are different from both those obtained when it is in either water, various hydrophobic solvents, or various other solvent compositions. Protonation of the bound adenine ring at the 3-position is offered as a possible explanation. Significant shifts are observed in both the stretching frequency of the carboxamide carbonyl of NAD+ and the rocking motion of the carboxamide NH2 group of NADH. These shifts are probably caused by hydrogen bonding with the enzyme. The interaction energies of these hydrogen-bonding patterns are discussed. The aromatic nature of the nicotinamide moiety of NAD+ appears to be unchanged upon binding. Pronounced changes in the Raman spectrum of the nicotinamide moiety of NADH are observed upon binding; some of these changes are understood and discussed. Finally, these results are compared to analogous results that were recently reported for liver alcohol dehydrogenase [Chen et al. (1987) Biochemistry 26, 4776-4784]. In general, the coenzyme binding properties are found to be quite similar, but not identical, for the two enzymes.     

The specific binding activities of human urinary radioiodinated colony-stimulating factor-1 to various human tissue cells

1. Colony-stimulating factor (CSF-1) was isolated from a large volume of fresh normal human urine by 5 steps of purification and enrichment. 2. The purification factor is 100,000 fold and the purified compound exhibits a 2.16 x 10(7) U/mg of protein sp. act. 3. The isolated CSF-1 is a sialoglycoprotein with 41.5% of carbohydrate. The almost complete removal of this carbohydrate moiety (up to 91%) was achieved by incubation with trifluoromethane sulfonic acid. 4. The deglycosylated CSF-1 (DG-CSF-1) possesses an apparent Mr 38,000 compared to native CSF-1 with an initial Mr 57,000 (Goa et al., 1988). 5. The features of the interaction of radio-iodinated [125I]CSF-1 with single cell suspensions from various human tissues (bone marrow, spleen, blood, peritoneal cavity, alveolar lavage, lymph node and thymus), were studied. 6. The binding activity of peritoneal macrophages was the highest among the cells examined and erythrocytes, thymus and blood granulocytes showed no CSF-1 binding. 7. On incubation with [125I]CSF-1 at 0 degrees C, cellular binding of [125I]CSF-1 reached a stable maximum within 16 hr. This is in contrast to the association behaviour at higher temperature. 8. At 37 degrees C, cellular associated [125I]CSF-1 levels reached, within 90 min, an unstable maximum which was up to 10 times less than that occurring under the same conditions at 0 degree C. From the Scatchard plot analysis, we obtained the affinity constant and the number of receptor(s). 9. The binding site is sensitive to trypsin. 10. The receptor alone, (labelled by cross-linking to [125I]CSF-1 with di-succinylimidyl-suberate), is a polypeptide with an approx. Mr 110,000. 11. Our results showed that the receptor of CSF-1 is a tyrosin-kinase.