Coding sequence cartridges for aminoglycoside phosphotransferase (APT) were isolated from bacterial transposon Tn903. When incorporated into a heterologous gene construction utilising the PGK1 promoter and terminator, the heterologous APT gene provided a G418-resistance determinant that functioned efficiently as a dominant marker for yeast in both multiple- and single-copy. Transformant colonies on selective medium appeared rapidly, within 36-48 h, and growth rate of the transformed cells was normal. A simple and highly sensitive radiolabelling assay for APT enzyme activity was developed for use with crude cell protein extracts. Enzyme activity units were equated to the amount of APT protein present in the cells, and the APT protein was shown to be stable in yeast. Heterologous APT expression was 130-fold reduced compared with homologous PGK1. This resulted from an estimated two-fold decrease in mRNA level and a 65-fold decrease in translation efficiency. The latter was unaffected by AUG sequence context change, but corresponded with a high frequency of minor codons in the APT-coding sequence. APT can be used as a semi-quantitative reporter of gene expression, whose useful features are in vivo detection via the G418-resistance phenotype and powerful cell-free assay.
HLA-A2 and -A3 genes have been modified in their third exon (second domain) by using in vivo recombination. In this method Escherichia coli are transfected with a plasmid which contains two highly homologous sequences (e.g., the third exons of HLA-A2 and -A3) and has been linearized by cleavage between these two sequences. Circularization takes place in the bacteria by homologous recombination leading to hybrid A2-A3 sequences. The analysis by DNA sequencing of a number of such recombinants shows that they indeed occur by homologous recombination (no insertions or deletions) and that the probability of crossing over decreases as the distance from the free end of DNA in the homologous region increases. No double recombinants were observed. These hybrid exons were reinserted into either HLA-A2 or HLA-A3 genes, thus generating a panel of functional hybrid genes containing one or several HLA-A2 specific substitutions in an HLA-A3 background or vice versa. These genes were expressed by transfection into murine P815-high transfection efficiency recipient cells. Serologic analysis leads to the conclusion that expression of polymorphic antigenic determinants specific for HLA-A2 (detected with M58, A2A28M1, and CR11.351 mAb) is linked to the presence of threonine residue (amino acid (AA) 142) and/or histidine residue (AA 145) and valine residue (AA 152). The expression of specific HLA-A3 polymorphic determinants (recognized by GAP-A3 mAb) is correlated with the existence of a asparagine residue (AA 127) and a aspartic residue (AA 161). But aspartic residue 161 contributes with glutamic acid residue 152 in the formation of the A3 epitope recognized by the anti-A3 mAb X1.23.2.
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