TABLE 2.
Bacterial plasmids constructed in this study
| Plasmid | Descriptiona | Constructionb |
||
|---|---|---|---|---|
| Vector | Insert | Digestion/oligonucleotides (5′-3′) | ||
| Recombination substrates | ||||
| pCIG1110 | oriT1(63-330mut)† | pCIG1028 | pCMS13 | AACTCTAGAACCCAATGCGCATAGCG |
| AACAAGCTTCCTCTCCCGTAGTGTTAC | ||||
| pCIG1116 | oriT1 HuX (15+3) (−7)‡ | pCIG1028 | Human DNA | CCATCTAGATTAGACACAGGCTCTACTCACACAG |
| TACAAGCTTAAAAATTCAACACAGCCTCTAAGTG | ||||
| pCIG1117 | oriT1 Hu5 (15+3) (−10)‡ | pCIG1028 | Human DNA | CCATCTAGATCTAAGATGCAGTAAGATCCCAGAC |
| TACAAGCTTTCAAGATTAGTGAGCAAGAAATGTG | ||||
| pCIG1122 | oriT1 Hu18 (10+2)‡ | pCIG1028 | Human DNA | CCATCTAGACCTTGAACCTATTCTGCCCATA |
| TACAAGCTTTCAAGGCTCTTGATGTTTGAGA | ||||
| pCIG1126 | oriT1 Hu5b (13+1)‡ | pCIG1028 | Human DNA | CCATCTAGAAGCTATGCACAACAGCATGG |
| TACAAGCTTAATCCCAATATTTGACCACCA | ||||
| pCIG1127 | oriT1 Hu7 (12+3)‡ | pCIG1028 | Human DNA | CCATCTAGACCTGGCGATAGAGCAAGACT |
| TACAAGCTTCTGACCACCTGCTCCAAAAT | ||||
| Plasmids used for intragenic complementation assays | ||||
| pCIG1070 | pSU19::TrwC (N450) | pSU19 | pCIG1051 | XbaI/BamHI |
| pCIG1086 | pSU19::TrwC | pSU19 | pSU1621 | XbaI/BamHI |
| pCIG1103 | pSU19::TrwC (N600) | pSU19 | pCIG1099 | XbaI/BamHI |
| pLA35 | pSU19::TrwC (N293) | pSU19 | pSU1600 | XbaI/BamHI |
| Plasmids used for yeast nuclear import/export assays | ||||
| pCIG1133 | pNIA::TrwC (N600) | pNIA3b | pSU1621 | ACCGGATCCCGATGCTCAGTCACATGGT |
| CCAGAATTCACTCGATGGCCTTGGTTTG | ||||
| pCMS2 | pNIA::TrwC (N293) | pNIA3b | pSU1621 | ACCGGATCCCGATGCTCAGTCACATGGT |
| ACCGAATTCAGCTGAAATCTATGCCGAG | ||||
| pCMS5 | pNIA::TrwC-NLS | pNIA3b | pSU1621 | ACCGGATCCCGATGCTCAGTCACATGGT |
| CCAGAATTCTACCTACCTTTCTTTCCGGCCTCCATGCC | ||||
| pCMS9 | pNIA::TrwC | pNIA3b | pSU1621 | ACCGGATCCCGATGCTCAGTCACATGGT |
| ACCGAATTCACCTTCCGGCCTCCATGCC | ||||
| pCMS10 | pNIA::TrwC (C774) | pNIA3b | pSU1621 | CCAGGATCCTTGGAGCCGTCTATAAC |
| ACCGAATTCACCTTCCGGCCTCCATGCC | ||||
| pCMS15 | pNEA::TrwC | pNEA3b | pCMS9 | ACCGGATCCCGATGCTCAGTCACATGGT |
| CCACTCGAGACCTTCCGGCCTCCATGCC | ||||
| pCMS16 | pNEA::TrwC (C774) | pNEA3b | pCMS10 | CCAGGATCCTTGGAGCCGTCTATAAC |
| CCACTCGAGACCTTCCGGCCTCCATGCC | ||||
| pLA44 | pNIA::TrwC*c | pCMS10 | pCMS9 | Mutagenic PCRd |
| GGCTGGCGGTTGGGGGTTA | ||||
| ACCGAATTCACCTTCCGGCCTCCATGCCGCG | ||||
| pLA66 | pNIA::TrwC (A904T) | pCMS10 | pLA44 | ACCGGATCCCGATGCTCAGTCACATGGT |
| ACCGAATTCACCTTCCGGCCTCCATGCC | ||||
| pMTX719 | pNIA::NLS(x5)-TrwC | pCMS9 | Oligose | GATCCCCAAGAAGAAACGGAAGGT |
| GATCACCTTCCGTTTCTTCTTGGG | ||||
| pMTX720 | pNIA::NLS(x2)-TrwC | pCMS9 | Oligos | GATCCCCAAGAAGAAACGGAAGGT |
| GATCACCTTCCGTTTCTTCTTGGG | ||||
| pMTX726 | pNIA::NLS-TrwC | pMTX719 | None | BamHI and religation |
| Plasmids used for immunofluorescence analyses | ||||
| pLA14 | pCEFL::TrwC | pCEFL | pET29:trwAC | ACCAAAGCTTATGCTCAGTCACATGGTATT |
| ACCAGGATCCTTACCTTCCGGCCTCCA | ||||
| pLA27 | pCEFL::TrwC (N293) | pCEFL | pCMS3 | BamHI/EcoRI |
| pLA28 | pCEFL::TrwC (N600) | pCEFL | pCIG1133 | BamHI/EcoRI |
| pLA29 | pCEFL::TrwC (C774) | pCEFL | pCMS10 | BamHI/EcoRI |
†, mutation affecting the nic site (TCT/A-to-GAG/A change); ‡, human sequences in the indicated human chromosomes (HuX, -5, -18, or -7). The “n+n′” format indicates the extent of the consensus sequence around the nic site; variations from consensus are indicated in parentheses (see the text for details and nomenclature).
The first column (Vector) lists the vector plasmids, the second column (Insert) lists the plasmids from which the inserts were obtained, and the third column (Digestion/oligonucleotides) indicates either the restriction enzymes used for cloning or the oligonucleotides used for PCR amplification of the desired fragment, with the restriction sites underlined.
TrwC*, TrwC mutant obtained by random mutagenesis. It carries missense mutation A904T and the additional peptide KVNSCSHGSSRSTRD after residue 964 of TrwC.
This PCR was performed under mutagenic conditions, as described in Materials and Methods. The PCR fragment was digested with BamHI and EcoRI and ligated into the same sites of the pCMS10 backbone.
Oligos, oligonucleotides. NLS sequences were added to the N terminus of TrwC by oligonucleotide hybridization and insertion at BamHI site of pCMS9.