Chromosomal DNA fragments as well as pUB110 plasmid DNA were entrapped in liposomes and transformation of $\underline{Bacillus}$ $\underline{subtilis}$ protoplasts with the entrapped DNA was tried for the purpose of increasing transformation efficiency.
$\underline{B}.{\underline$subtilis}$ chromosomal DNA fragments of 10-15 megadalton sizes were encapsulated in large unilamellar vesicles (LUV) using ether injection method. The same injection method was used for encapsulating plasmid pUB110 into the LUV. The liposome containing DNA were then tried to fuse with $\underline{B}.$\underline{subtilis}$ protoplasts under the presence of polyethyleneglycol (PEG) as usual.
The formation of LUV having 0.1μm average diameter was confirmed by election microscopy. The average weight of the LUV was found to be more than 2 megadaltons.
The $^3H$-labeled chromosomal DNA of $\underline{B}.$\underline{subtilis}$ was entrapped in liposomes and the liposome suspension was applied to Sepharose CL-4B column. The entrapment of chromosomal DNA in the liposomes was proved by coincident peaks of radioactivity and optical density at fraction number 15. About 5μl of chromosomal DNA was entrapped in 1μmol of liposome.
Intact cells of the two tryptophan requiring mutants of $＼underline{B}$.$＼underline{subtilis}$ 168 and BD170, were found to mutate reversely to non-requiring strains with frequency in the order of $10^{-6}$. This frequency, however, increased by 100-fold when the cells were protoplasted. The mechanism involved is remained to be studied.
Entrapment of plasmid pUB110 DNA into liposome was also confirmed by the agarose gel electrophoresis. The transformation of $\underline{B}.$\underline$subtilis}$ protoplasts with liposome-entrapped plasmid DNA was succeeded, but the frequency was about 10-fold lower than that of PEG-induced protoplast transformation.
일반적인 DNA 전달기작-즉 Transformation and Transfection 이 아닌 DNA 를 포획한 라이포솜을 DNA 전달매체로 사용한 형질변환실험을 수행하였다. 에테르 주입방법에 의해 만들어진 라이포솜을 전자현미경으로 관찰한 결과 단일층 확인되었고 이 라이포솜안으로 $\underline{B}$.$\underline{subtilis}$ 염색체 조각이 포획된 사실을 Sepharose CL-4B크로마토그라피로 확인하였다. $\underline{B}$.$\underline{subtilis}$ 프로토프라스트의 높은 역 돌연변이빈도 때문에 염색체 조각을 포획한 라이포솜에 의해서는 형질변환실험을 할 수 없었다. 또, 라이포솜안으로 pUB110플라스미드가 원상태로 생물학적 활성도를 유지한채 포획된 사실을 알았고 이 플라스미드를 포획한 라이포솜과 프로토프라스트를 PEG에 의해 세포막 융합을 시켜 형질변환실험을 한 결과 1.06 × $10^{-4}$ 형질변환체수＼㎍ DNA or 2.98 × $10^{-3}$ 형질변환체수/재생 체수의 형질변환체가 발생하였다.
LUV 라이포솜이 프로토프라스트의 재생율을 4배에서 최고 18배 증가시키는 사실로 보아 라이포솜이 프로토프라스트에 손상을 주는 대신 상호작용하여 재생에 좋은 효과를 미치는 것을 알 수 있었다.
