Mutagenic Primers (sense strand)


 A

 bcys20aser
 5' CTG -TTC G-TT CTG -TTC TCC ATG AAG TAC GTT TGG CCG CC 3'
 cyseser -SnaB1


 bgly434cys
 5' GAA A-TT GCT GAC TGC C-TT GCT TCC GCA GAA CGA GCA CAT AAG 3'
 gly~cys -Bspl285I


 bser844cys
 5' GCG AAC AAA CGC CGC TGC CAG A-TT CTC GAC GAA GC 3'
 ser~cys -Xbal

 B
 cys644ser
 5' GCC GAG TCG -TTT ATC GCA G-TT TCT GGT GAG CAA CTG GAC 3'
 cyseser



 cysl400ser +EcoR1
 5' CGT CTG TCA CGC AAA GTT AAG CTG AAT TCG
 cyseser
 AAA ATT GAT AAG TCT GTA ATG GCA GG 3'
 -Clal

 C
 cLys90 a ser
 5' GGC ATG AAA G-TT AAG TCT ACT GGC CGG ATC CTG GAA G 3'
 cysrser +BamH1

Figure 4-2. Oligonucleotides for cysteine mutagenesis of the unc operon. Shown are the
 sense strands of the mutagenic primer pairs. The desired mutations are shown
 in color. Bold script indicates a change in nucleotide. Restriction enzyme
 recognition sequences that were silently added or abolished are underlined.
 Mutations were introduced as described in the Materials and Methods. A)
 Blue indicates the cysteine mutations constructed in uncF(b). The SnaBI,
 Bspl285I and Xbal restriction sequences were knocked out along with the
 bcys200ser, bgly434cys and bser844cys, TOSpectively, to facilitate screening. B) Red
 indicates the cysteine mutations generated in uncH(G). The Clal sequence
 was knocked out and the EcoRI site was generated along with Scysl400ser foT
 screening purposes. C) Green indicates the cysteine mutation made in unc(u).
 The BamnHI sequence was added to screen for the acys90ser mutation.