|Budget Amount *help
¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1991 : ¥300,000 (Direct Cost : ¥300,000)
Fiscal Year 1990 : ¥1,400,000 (Direct Cost : ¥1,400,000)
To block the progression of the DNA replication fork in E. coli cells, at least two factors are required ; one is the specific terminus (ter) sequence (-22bp) and the other is ter-binding protein. (1) To investigate behavior of an E. coli genome carrying the region unreplicated after DNA synthesis period and effects this genome have on host cells, we did the following experiments ; an E. coli strain, the genome of which carried a region flanked by two ter sequences, was constructed from a strain deficient in ter-binding protein. We expected that in the presence of ter-binding protein this strain would be lethal or would show poor growth due to the presence of an unreplicatable region on the genome. As expected, when the tau gene which codes for ter-binding protein was introduced into the E. coli strain, growth rate of the strain was greatly reduced, in comparison with that of the control strain. This suggested that blockage against' the DNA replication fork at the ter site is leaky. A
greater inhibition for the replication fork at the ter site is needed to assess the fate of the genome with an unreplicatable region. (2) To determine whether or not E. coli ter system is functional on eucaryotic DNA replication, we investigated the DNA synthesis of SV40 DNA carrying the ter sequence, in both crude and purified enzyme in vitro, in the presence of ter-binding protein. As well as in the E. coli in vitro DNA replication system, blockage of the DNA replication fork at the ter site was evident under both conditions. The ter sequence-ter binding protein complex could impede the helicase action of SV40 large T antigen, in a polar fashion. A similar activity was observed previously when we used 3 types of E. coli helicases.
大腸菌のゲノム上のlac遣伝子内に、人工的に複製終結点を導入し、その方向性から、少なくともゲノム上のlacとtrp遺伝子間の領域が複製不能あるいは、著しく遅れると予想される株を作り調べた。終結タンパク質の生産をコントロ-ルすることで、未複製領域がない状態から、ある状態へ変化したところ、明らかに菌の増殖が遅くなることが判明した。しかし致死に至らぬこと、又その効果が菌株によって異なることも判明した。一方、大腸菌の終結システムが、真核生物においても有効か否かを調べ。酵母においては、予備的ではあるもののin vivoで有効であること、さらにSV40の試験管内DNA複製系を用いた実験では、ヒト細胞抽出液のDNA複製活性を、大腸菌の場合と同様、終結点と終結タンパク質の複合体が、極性をもって阻害することを見出した。 Less