TABLE 2.
Function and name | Sequencea | Locationb | Utilization |
---|---|---|---|
Construction of mutants | |||
gutbc1 | CTGCCATACCAGCAATGGC | −285 | Cloning gutB with an internal 480-bp deletion |
gutbc2 | TGCCAGCGGACCAGACTTC | +315 | |
gutbc3 | TATCGTCATCGGCTTTATCTG | +796 | |
gutbc4 | ATCCTGCGAGCTCAGGTG | +1455 | |
gutF1 | GCAAGTAAAGGCATC | −350 | Cloning part of gutR to create a frameshift at |
gutR1 | TGGTGAATCGTCCATCAAG | +689 | PstI restriction site (codon 48) |
gutR2 | TGATGCTGGCAGAATCATC | −451 | Cloning gutM to create a frameshift at PstI |
gutC1 | TGGAACAGCTTCATGAAGC | +574 | restriction site (codon 16) |
Complementation of mutants | |||
gutRBglII | CATAAGATCTGGGAGGTAACCTG | −24 | Cloning gutR in pT1NX |
gutRSpeI | TATCAACTAGTTCATTTAAAGTCCCTC | +1884 | |
gutMBglII | CCTTAACAGATCTCACGGAGGGAC | −29 | Cloning gutM in pT1NX |
gutMSpeI | CATCTACTAGTTTTAAATTGTATTC | +522 | |
Transcription analysis | |||
ribu1 | TATTATGGGCAGGATAATGC | −302 | DNA probe (the primer location is indicated |
gutF3 | GCCGTTAACAACCGTAGCG | +117 | with respect to guF) |
gut3 | GACTTTAAGGACCCTAA | +133 | RT-PCR analysis (the primer location is |
gutbc4 | ATCCTGCGAGCTCAGGTG | +5277 | indicated with respect to guF) |
gut5 | TTTTCAACTTCGTCAGGATC | +197 | Identification of the transcription start site of |
gutF3 | GCCGTTAACAACCGTAGCG | +117 | the gut operon (the primer location is indicated with respect to guF) |
Overexpression of genes | |||
gutRBamHI | GGGAGGGATCCGGATTGAATAG | −14 | Cloning gutR in pQE30 |
gutRSalI | CAAAACGTCGACTTAAAGTCCC | +1881 | |
gutMBamHI | CGGAGGGATCCAAAATGAACG | −14 | Cloning gutM in pQE30 |
gutMHindIII | CTCCTAAGCTTAAATTGTATTC | +519 | |
Gel mobility shift and DNase I footprinting | |||
gut8D | GTCAAGCACCGCTAACC | −200 | Amplified 179-bp DNA fragment of the gut |
gutF4 | TTTCTGCTCTATGGTAGC | −22 | promoter region (the primer location is indicated with respect to guF) |
Letters in italics indicate newly created restriction sites.
The numbers indicate the position of the 5′ end of the primer with respect to the start codon of the gene specified in the “Utilization” column.