Transfection of wild-type and 'Fanconi anemia-like' mouse lymphoma mutant cells by electroporation

An electroporation protocol for the successful transfection of mouse lymphoblastoid cells has been developed. Two cell lines, a normal and a mutant sensitive to DNA cross-linking agents, were used. The optimum conditions of electroporation in terms of uptake of the fluorescent dye lucifer yellow coupled with low toxicity were established. Subsequently, these conditions were used to achieve stable transfection by a plasmid expression vector. The plasmid integration patterns were determined by Southern blot analysis.     

Abnormal lymphokine production: a novel feature of the genetic disease Fanconi anemia

Summary The correction of chromosomal hypersensitivity to mitomycin C (MMC) in Fanconi anemia (FA) human lymphoblasts is observed by growth in a medium conditioned by normal human cells. Under the same conditions, the cytotoxic effect of MMC on FA cells is restored to an almost normal level. The addition of interleukin-6 (IL-6) to an unconditioned culture medium increased the resistance of FA cells to MMC cytotoxicity. This correcting effect is partially abolished by addition of an anti-IL-6 antibody to the conditioned medium. Both lymphoblasts and fibroblasts derived from FA patients demonstrate a reduction in IL-6 production. Moreover, this lymphokine is not induced by tumor necrosis factors and (TNF and TNF) in FA cells, as is the case in normal cells. It is suggested that the observed deficiency in IL-6 production may account for one of the major characteristics of FA disease, i.e., the defect in differentiation of the hematopoietic system.     

A yeast protein analogous to Escherichia coli RecA protein whose cellular level is enhanced after UV irradiation

Summary In Saccharomyces cerevisiae, a protein was recognized by polyclonal antibodies raised against homogeneous Escherichia coli K12 RecA protein. The cellular level of the yeast protein called RecAsc (molecular weight 44 kDa, pI 6.3), was transiently enhanced after UV irradiation. Protease inhibitors were required to minimize degradation of the RecAsc protein during cell lysis. The RecAsc protein exhibited similar basal levels and similar kinetics of increase after UV irradiation in DNA-repair proficient (RAD ⁺) strains carrying mitochondrial DNA or not (rho ⁰). This was also true for the following DNA-repair deficient (rad ^-) strains: rad2-6 rad6-1 rad52-1, a triple mutant blocked in three major repair pathways; rad6-, a mutant containing an integrative deletion in a gene playing a central role in mutagenesis; pso2-1, a mutant that exhibits a reduced rate of mutagenesis and recombination after exposure to DNA cross-linking agents.     

Migration of peripheral T and B cells into the thymus of aging (NZB × SJL)F1 female mice

Aging NZB × SJL (NS) female mice provide a unique model of thymopathology characterized by the intrathymic accumulation of large numbers of mature T and B cells. The purpose of the present work was to examine the possibility that this phenomenon results from the invasion of the thymus by cells from the periphery. Lymphoid cells labeled with chromium-51 or indium-111 were injected into syngeneic recipients to study their patterns of in vivo migration. Lymph node (LN) or spleen cells were found to localize significantly (1–2% of injected radioactivity) into the thymus of 12-month-old NS females but not into that of young recipients or of old NS males. However, intrathymic localization of injected LN cells was observed in castrated NS males which exhibit the same thymopathology as NS females. Both radiolabeled T and B cells were found to enter the thymus of aged NS females but the latter cells about three times less efficiently than the former. Moreover, while thymocytes from young NS females were unable to recirculate to LN, those of old NS females showed increased LN-seeking capacity and part (1%) of them did migrate back into the thymus of old but not young NS females. In additional cell transfer experiments, the intrathymic migration of B cells into old NS females was further documented by using the antibody response to sheep erythrocytes as a tracer. Taken together, these observations indicate that the thymus of aging NS female mice is permeable to recirculating lymphocytes, suggesting that at least part of the mature T and B cells detected in this thymus, are migrants from the periphery.     

Additional Restriction Endonuclease Cleavage Sites on the Bacteriophage P22 Genome

We present complete restriction endonuclease cleavage site maps of the bacteriophage P22 chromosome for 16 enzymes with six base recognition sequences, thereby positioning 116 new sites on the chromosome. Twenty-four such restriction maps for P22 DNA, containing 162 sites, have now been completed, and three enzymes were found that did not cut P22 DNA. Our results are consistent with the ideas that ClaI does not cleave the methylated recognition sequence ATCGA(me)T or A(me)TCGAT and StuI does not cleave the methylated recognition sequence AGGCC(me)T.     

Localization of Two Functions of the Phosphoribosyl Anthranilate Transferase of Escherichia coli to Distinct Regions of the Polypeptide Chain

The trpD gene specifies a polypeptide which has both glutamine amidotransferase and phosphoribosyl anthranilate (PRA) transferase activities. Deletions fusing segments of trpD to the gene preceding it in the operon, trpE, were selected in strains carrying various trpD point mutations. The selection procedure required both that a deletion enter trpE and that it restore the PRA transferase activity which the parent trpD point mutant lacked. Deletion mutants were found which had PRA transferase activity although the first third of trpD was deleted. The existence of the mutants proves that a terminal segment of trpD is sufficient to specify a polypeptide having PRA transferase activity. The location of the deletion end points on the genetic map of trpD defines the extent of the trpD segment required for PRA transferase activity. This segment did not overlap the initial region of trpD required to specify the glutamine amidotransferase function of the trpD polypeptide. These results support the hypothesis (M. Grieshaber and R. Bauerle, 1972; H. Zalkin and L. H. Hwang, 1971) that the bifunctional trpD polypeptide might have evolved by fusion of a gene specifying a glutamine amidotransferase with a gene directing PRA transferase synthesis.