Induction of Antibiotic Resistance in Paramecium tetraurelia by the Bacterial Gene APH-3'-II

We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neo^r) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neo^r ORF was inserted, the plasmid was referred to as pPXV-NEO. Delivery of approximately 10–20 picoliters of linearized PXV-NEO at > 2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neo^r-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G-418, and the concentrations of neo^r-specific DNA and neo^r-specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.     

Activation of Host Lymphocytes cultured with Cancer Cells treated with Neuraminidase

IMMUNE response to malignancy can be quantified by measurement of blastic transformation of host lymphocytes cultured with malignant target cells in which the capacity to synthesize polynucleotides has been blocked^1–3. We have used this type of target cell culture to study the effects of pretreatment of malignant target cells with agents intended to accentuate host immune response. As an initial study we have evaluated the action of the Vibrio cholerae enzyme, neuraminidase, which removes sialic acid groups from the cancer cell membrane. There is evidence that removal of such saccharide radicals from the cell surface of experimental tumours facilitates immune recognition of antigens associated with the tumour cells^4–6. We have observed an accentuated lymphoblastic response of rat host peripheral blood leucocytes cultured with Novikoif hepatoma target cells pretreated with neuraminidase. Eight out of eleven humans studied have shown a similar response to enzyme treated malignant cells.     

Haemoglobin Miyada,a β—δ Fusion Peptide (Anti-Lepore) Type discovered in a Japanese Family

SEVERAL slow-moving abnormal haemoglobins associated with thalassaemia-like stigmata have been encountered during the past 13 yr^1–6. The non-α-chains of these haemoglobins are the fusion products⁷ of δ and β-chains; the N-terminal end containing a part of the β-chain joined to a fragment of the β -chain ending in the C-terminal. But no structural variants of anti-Lepore type (fused in reverse β-δ) have yet been demonstrated. In a systematic screening survey for abnormal haemoglobins⁸, we discovered a new variant named Hb Miyada which may possess a fused non-α-chain.