Site specific mutagenesis: insertion of single noncomplementary nucleotides at specified sites by error-directed DNA polymerization

We have utilized infidelity of DNA synthesis as a basis for site-directed mutagenesis. Both an endonuclease restriction fragment and a synthetic oligonucleotide were used as primers. DNA polymerase from bacteriophage T4 was used to elongate primer termini to a position immediately adjacent to two different preselected positions on phiX174 DNA templates. Then, the error-prone DNA polymerase from avian myeloblastosis virus was used to insert single non-complementary nucleotides at the designated positions at high efficiency. DNA sequence analysis confirmed that the mutant phage produced as a result of each site-specific mutagenesis reaction contained the nucleotide that was complementary to the one provided during the DNA copying reaction. The general applicability of this methodology to cloned DNAs will be discussed.     

A Thick-Walled Organism Isolated from the Cockroach Gut by Using a Spent Medium Technique

By using a conditioning technique whereby complex media are inoculated several times with bacteria from the hindgut of the cockroach Eublaberus posticus, a succession of bacterial types occurred. An obligately anaerobic, pleomorphic, thick-walled, gram-positive organism is described which was isolated by this culturing technique.     

2,4-D-Induced Changes in the K+ Uptake of Wheat Roots at Different pH Values

The effects of an auxin herbicide, 2,4-D, at a concentration of 0.01 mM, on the K⁺ uptake and efflux of excised roots of wheat (Triticum aestivum L. cv. Rannaya) were investigated at different pH values. The K⁺ movement was monitored with a K⁺ (⁸⁶Rb) tracer. In parallel experiments the ATPase activities of microsomal fractions were determined by the inorganic phosphate liberation method. 2,4-D inhibited the K⁺ uptake especially at low pH, irrespective of whether Ca²⁺ was present or not. No marked changes were observed in the K⁺ efflux properties at pH values above 4. The inhibitory effect on K⁺ uptake exhibited a correlation with the hydrocarbon solubility of the herbicide, but not with the 2,4-D-induced decrease of the ATPase activity. It is suggested that 2,4-D exerts a non-specific effect on the lipid-protein interactions, giving rise to a generalized alteration of the transport barrier properties of the plasma membrane even at as low a concentration as 0.01 mM.     

Phosphorylation of the lymphoid cell kinase p56lck is stimulated by micromolar concentrations of Zn2+

In particulate fractions from LSTRA lymphoma cells, tyrosine phosphorylation of the lymphoid specific tyrosine kinase p56lck is elicited by Zn2+ in the absence of other divalent cations. Zn2+ alone also induces autophosphorylation of immunoprecipitated p56lck. The effect of Zn2+ is dose dependent; it is detected at concentrations of Zn2+ as low as 5 microM and reaches a maximum at 100 microM Zn2+. Among other divalent cations tested, Mn2+, and Co2+ to a lesser extent, were also effective. Zn2+ also stimulated p56lck phosphorylation in the presence of Mg2+ ions at physiological concentration, whereas orthovanadate had no effect. These results suggest that Zn2+ activates the autophosphorylation of p56lck; this fact could be related with the stimulating effect of Zn2+ in the activation of T lymphocytes.     

DNA damage and repair in brain: relationship to aging.

The usefulness of conducting DNA damage and repair studies in a postmitotic tissue like brain is emphasized. We review studies that use brain as a tissue to test the validity of the DNA damage and repair hypothesis of aging. As far as the accumulation of age dependent DNA damage is concerned, the data appear to overwhelmingly support the hypothesis. However, attempts to demonstrate a decline in DNA repair capacity as a function of age are conflicting and equally divided. Possible reasons for this discrepancy are discussed. It is suggested that assessment of the repair capacity of neurons with respect to a specific type of damage in a specific gene might yield more definitive answers regarding the role of DNA repair potential in the aging process and as a longevity assurance system.     

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.