Characterization of the 5'' to 3'' exonuclease associated with Thermus aquaticus DNA polymerase.

Thermus aquaticus DNA polymerase was shown to contain an associated 5' to 3' exonuclease activity. Both polymerase and exonuclease activities cosedimented with a molecular weight of 72,000 during sucrose gradient centrifugation. Using a novel in situ activity gel procedure to simultaneously detect these two activities, we observed both DNA polymerase and exonuclease in a single band following either nondenaturing or denaturing polyacrylamide gel electrophoresis: therefore, DNA polymerase and exonuclease activities reside in the same polypeptide. As determined by SDS-polyacrylamide gel electrophoresis this enzyme has an apparent molecular weight of 92,000. The exonuclease requires a divalent cation (MgCl2 or MnCl2), has a pH optimum of 9.0 and excises primarily deoxyribonucleoside 5'-monophosphate from double-stranded DNA. Neither heat denatured DNA nor the free oligonucleotide (24-mer) were efficient substrates for exonuclease activity. The rate of hydrolysis of a 5'-phosphorylated oligonucleotide (24-mer) annealed to M13mp2 DNA was about twofold faster than the same substrate containing a 5'-hydroxylated residue. Hydrolysis of a 5'-terminal residue from a nick was preferred threefold over the same 5'-end of duplex DNA. The 5' to 3' exonuclease activity appeared to function coordinately with the DNA polymerase to facilitate a nick translational DNA synthesis reaction.     

Glucocorticoids in muscular dystrophy: beneficial effects of dexamethasone on avian myopathy

A corticosteroid with mixed glucocorticoid-mineralocorticoid actions was previously shown to improve neuromuscular function in muscular dystrophic chickens. The significance of that finding was recently underscored by reports that a mixed-action corticosteroid improved muscle function in Duchenne dystrophy patients, albeit at high doses. In the present study a pure glucocorticoid improved function and retarded muscle histopathology in the chicken, but a pure mineralocorticoid did not. These observations suggest that elucidation of mechanisms by which glucocorticoids beneficially affect dystrophic muscle could lead to development of more effective therapies.     

High-level conjugation of chelating agents onto immunoglobulins: use of an intermediary poly(L-lysine)-diethylenetriaminepentaacetic acid carrier

Diethylenetriaminepentaacetic acid (DTPA), a strong chelating agent, was covalently linked to murine monoclonal anti-HLA IgG1 antibody (H-1) with the use of poly(L-lysine) (Mr 14,000) as a multivalent, intermediary carrier, via thiol-disulfide exchange reaction. The conjugates contained up to 42.5 mol DTPA per mol antibody, and retained over 90% of their antibody activity in vitro. The conjugates incorporated gadolinium (Gd) through an exchange reaction with Gd-EDTA, used to prevent colloid formation and nonspecific binding of the free metal. The IgG-poly(L-lysine)-DTPA-Gd had a greater effect per mol on proton relaxation rates than DTPA-Gd itself. Use of poly(L-lysine) as an intermediary carrier for attachment of chelating agents to IgG thus offers great potential for achieving high-specific-activity conjugates, particularly for use as biologically specific contrast agents in nuclear magnetic resonance imaging.     

Serotonin immunoreactivity in the nervous system of the dragonfly nymph

Serotonin-like immunoreactivity was mapped using an antiserotonin antibody in wholemounts of the ventral nerve cord from dragonfly nymphs (Epitheca sp. and Pachydiplax longipennis). In both species, an immunoreactive cell ventral to each connective tract and an immunoreactive median cell cluster on the ganglion ventral surface were found in the unfused abdominal ganglia. Axon(s) from the median cell cluster branch in the anterior unpaired median nerve. Posterolaterally, in all of the ganglia examined, two or more intensely immunoreactive, bilaterally symmetric pairs of neurons were seen. Comparison of these posterolateral neurons, which appear to be serially homologous, with similar antiserotonin immunoreactive neurons described in other insects suggests that these neuron pairs may have cross-species homology as well.     

Only aldehyde fuchsin made from pararosanilin stains pancreatic B cell granules and elastic fibers in unoxidized microsections: problems caused by mislabelling of certain basic fuchsins

The most distinctive property of aldehyde fuchsin is its staining of certain nonionic proteins and peptides in unoxidized cells and tissues. These substances include granules of pancreatic islet B cells, elastic fibers and hepatitis B surface antigen. Aldehyde fuchsin made from two different basic fuchsins, each certified by the Biological Stain Commission and labelled C.I. (Colour Index) No. 42500 (pararosanilin), did not stain pancreatic B cells at all. Stain Commission's records and retesting showed that each of the "faulty" basic fuchsins was not pararosanilin, but rosanilin, whose Colour Index number is 42510. These basic fuchsins were labelled with the wrong Colour Index number when packaged. Additional basic fuchsins were coded by V.M.E. and tested by R.W.M. for their capacity to make satisfactory aldehyde fuchsins. Only certain of these aldehyde fuchsins stained unoxidized pancreatic islet B cells. The same aldehyde fuchsins stained elastic fibers strongly. Each basic fuchsin whose aldehyde fuchsin was judged satisfactory proved to be pararosanilin. Aldehyde fuchsin solutions made from other basic fuchsins stained elastic fibers only weakly and did not stain pancreatic B cells at all in unoxidized sections. Each basic fuchsin whose aldehyde fuchsin was unsatisfactory proved to be rosanilin. It appears that only aldehyde fuchsin made from pararosanilin stains unoxidized pancreatic B cell granules dependably. We found that basic fuchsins from additional lots of Commission-certified pararosanilin and rosanilin were also labelled with incorrect Colour Index numbers when packaged. Steps were taken to prevent recurrences of such mislabelling which has made it difficult until now to correlate differences in the properties of pararosanilin and rosanilin. A table is provided of all basic fuchsins that have been certified by the Biological Stain Commission since 1963 when they began the practice of subdesignating basic fuchsins according to whether they are pararosanilins or nonpararosanilins. The consumer can readily determine from the certification number on the label the correct subdesignation of any Commission-certified basic fuchsin listed here. Until now, mislabelling of some lots of pararosanilin as rosanilin and vice-versa has confused and frustrated the users of basic fuchsins in other applications such as the carbol fuchsin staining of tubercle bacilli and certain cytochemical tests, e.g. esterase and acid phosphatase, that utilize hexazotized pararosanilin as a coupling reagent. Consumers experiencing trouble with any Commission-certified dye should look to the Biological Stain Commission for help. This is an important reason for purchasing, whenever possible, only Biological Stain Commission certified dyes.     

Properties of the 3' to 5' exonuclease associated with porcine liver DNA polymerase gamma. Substrate specificity, product analysis, inhibition, and kinetics of terminal excision.

Porcine liver DNA polymerase gamma was shown previously to copurify with an associated 3' to 5' exonuclease activity (Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988-995). The 3' to 5' exonuclease has now been characterized, and like the DNA polymerase activity, it has an absolute requirement for a divalent metal cation (Mg2+ or Mn2+), a relatively high NaCl and KCl optimum (150-200 mM), and an alkaline pH optimum between 7 and 10. The exonuclease has a 7.5-fold preference for single-stranded over double-stranded DNA, but it cannot excise 3'-terminal dideoxy-NMP residues from either substrate. Excision of 3'-terminally mismatched nucleotides was preferred approximately 5-fold over matched 3' termini, and the hydrolysis product from both was a deoxyribonucleoside 5'-monophosphate. The kinetics of 3'-terminal excision were measured at a single site on M13mp2 DNA for each of the 16 possible matched and mismatched primer.template combinations. As defined by the substrate specificity constant (Vmax/Km), each of the 12 mismatched substrates was preferred over the four matched substrates (A.T, T.A, C.G, G.C). Furthermore, the exonuclease could efficiently excise internally mismatched nucleotides up to 4 residues from the 3' end. DNA polymerase gamma was not found to possess detectable DNA primase, endonuclease, 5' to 3' exonuclease, RNase, or RNase H activities. The DNA polymerase and exonuclease activities exhibited dissimilar rates of heat inactivation and sensitivity to N-ethylmaleimide. After nondenaturing activity gel electrophoresis, the DNA polymerase and 3' to 5' exonuclease activities were partially resolved and detected in situ as separate species. A similar analysis on a denaturing activity gel identified catalytic polypeptides with molecular weights of 127,000, 60,000, and 32,000 which possessed only DNA polymerase gamma activity. Collectively, these results suggest that the polymerase and exonuclease activities reside in separate polypeptides, which could be derived from separate gene products or from proteolysis of a single gene product.