The tyrosine tRNAs specified by the E . coli amber suppressor gene su: , , and the wild type non-suppressing allele su,, were selectively labelled with 3 2 P in E . coli infected with transducing phage p80 carrying either of these genes. The determination of the nucleotide sequence of the S U :, , tyrosine tRNA is described. This tRNA has the anticodon CUA. The s u~, tRNA differs from su: , , tRNA by a single nucleotide and has the anticodon GUA where G is a modified guanylic acid residue. E . coli su-cells contain two tyrosine tRNAs (I and 11). The determination of the nucleotide sequence of these is described. Species I is identical to su,, tRNA while species I1 differs by two nucleotides in the variable loop region.
Escherichia coli HB101 harboring an expression plasmid that bears the calf prochymosin gene controlled by the tac promoter was cultivated under different conditions in order to find an optimal fermentation arrangement that would lead to maximal prochymosin yield. Our results indicate that it is advantageous to use lactose in the double role of inducer and carbon/energy source when foreign gene expression is controlled by the tac promoter and the gene product is only moderately toxic owing to its accumulation in the form of an intracellular body. Glucose, on the other hand, may be used when expression should be repressed. Growth temperature substantially influenced the specific rate of prochymosin and beta-lactamase gene expression and the plasmid copy number. Three phases were distinguished in the time course of the fermentation on lactose: exponential growth practically without prochymosin synthesis, linear growth with prochymosin synthesis, and prochymosin synthesis without growth of biomass. The synthesis of prochymosin in the form of intracellular inclusion body was accompanied by the loss of respiratory activity of the cell and the loss of its ability to multiply. Sixteen hours cultivation at 37 degrees C in a complex medium with lactose as inducer and carbon/energy source resulted in up to 30% of the volume and 48% of the total protein of biomass being accumulated for as prochymosin inclusion bodies. The concentration of extractable enzymatically active chymosin in the culture reached 12 mg/L.
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