Table 1.
Plasmids used in this study
| Plasmid | Description | Source or reference |
|---|---|---|
| pUC119 | pUC19 with M13 origin of replication | Sambrook et al., 1989 |
| pRB306 | TUB1 in pRB322 | Schatz et al., 1986b |
| pRB326 | TUB1 in YCp50 | Schatz et al., 1986b |
| pJJ217 | HIS3 in pUC18 polylinker | Jones and Prakash, 1990 |
| pJJ282 | LEU2 in pUC18 polylinker | Jones and Prakash, 1990 |
| pRS317 | LYS2 in pRSS56 | Sikorski and Hieter, 1989 |
| pRB2063 | TUB1 fragment (SphI–SacI) from pRB306 inserted into SphI–SacI-cut polylinker of pUC119 | This study |
| pRB2065 | BamHI–SalI fragment from pJJ282 (LEU2 gene) Klenow filled and inserted into the HpaI site of pRB2063, downstream of TUB1; LEU2 is oriented in the same direction as TUB1 | This study |
| pRB2067 | SalI–KpnI (partial) fragment from pJJ217 containing the entire HIS3 gene and flanking regions, inserted into XhoI–KpnI vector fragment from pRB2063; HIS3 is in the same orientation that TUB1 was; note that this disruption construct removes the first 763 bp of the 1580-bp ORF downstream of TUB1 (YML086c) | This study |
| pRB2070 | PvuII fragment from pRS317 containing LYS2 gene inserted into HpaI site of pRB2063; LYS2 is oriented in the same direction as TUB1 | This study |
| pRB2666 | BglII fragment containing TUB3 from pRB300 ligated into BamHI site of pUC119 | This study |
| pRB2668 | SalI–SmaI containing HIS3 fragment from pJJ217 ligated into XhoI–NdeI (Klenow-filled) vector fragment from pRB2666; HIS3 is in the same orientation as TUB3 was | This study |
| pRB2254 through pRB2412 | pRB2065 mutagenized via site-directed mutagenesis, yielding three isolates of each Ala-scan mutation, tub1-801 through tub1-853 | This study |