To amplify XBP1 mRNA (NM_005080), PCR was performed for 35 cycles (95°C for 30s 58°CC for 30s 72°CC for 1 min) using the PCR primers 5'-CTG GAA AGC AAG TGG TAG and' and 5'-CTG GGT CCT TCT GGG TAG AC-3' with AmpliTag Cold DNA polymerase (#N808-0241; Applied Biosystems).
Approximately 200 ng DNA was used as template for a 15-cycle cold PCR amplification using KOD polymerase (TaKaRa).
To explore whether also OsPP2C genes from subfamily A play functional roles in ABA mediated signaling pathways and stress responses, we analyzed the expression of 10 OsPP2C genes in rice plants treated with ABA, salt, osmotic (mannitol) and cold stress by reverse transcriptase polymerase chain reaction (RT-PCR).
To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5′-to-3′ exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E DNA ddNTP) and binary (E DNA) complexes.
These results not only explain a cold-sensitive phenotype for a mutant polymerase but also demonstrate large, long-range effects from a conservative mutation.
Four diploid inbred B. distachyon lines, Bd3-1, Bd21-1, Bd1-1, and Bd29-1 were used to characterize cold induced IRIP gene expression by quantitative real-time polymerase chain reaction (qRT-PCR).
This was achieved by using an E. coli strain that carries a cold-sensitive mutation in the 5′ 3′ exonuclease domain of DNA polymerase I, which is lethal at temperatures below 20°CC.
To determine how the I707L mutation causes a cold-sensitive phenotype, we co-crystallized the mutant KlenTaq1 DNA polymerase with DNA and a substrate nucleotide using similar procedures as those for wild-type KlenTaq1.
We thereby recovered RPO26 (encoding a shared subunit of all three nuclear RNA polymerases) and RPO31 (encoding the largest subunit of RNA polymerase III) as moderate and weak suppressors of tgs1 ∆ cold sensitivity, respectively.
It is thus remarkable that mutation from isoleucine to leucine at position 707 in DNA polymerase I from Thermus aquaticus resulted in an unusual cold-sensitive phenotype.
