An attempt was made to clone an allosteric lactate dehydrogenase (LDH) gene of $\underline{Lactobacillus}$ $\underline{casei}$ in $\underline{Escherichia}$ $\underline{coli}$. The chromosomal DNA of $\underline{L.}$ $\underline{casei}$ was restricted and hybridized with a synthetic probe. Plasmid pUC9 was used as a vector.
A mixture of oligonucleotides was synthesized by modified phosphite triester method based on the nucleotide sequence of the LDH produced by $\underline{L.}$ $\underline{casei}$. The synthesized probe was composed of 17 bases and was labeled with [$\gamma-^{32}P$] ATP before use. The chromosomal DNA of $\underline{L.}$ $\underline{casei}$ was digested with StuI and then hybridized with the labeled probe following the procedures of Southern hybridization. The probe hybridized to a 2.3 killobase (kb) chromosomal DNA fragment. Again the chromosomal DNA of $\underline{L.}$ $\underline{casei}$ was digested with StuI and DNA fragments of 2.3 kb size were isolated. The isolated DNA fragments were ligated to SmaI site of pUC9. With the ligation mixture E. coli JM83 was transformed to make a gene library.
Approximately 1500 $\underline{E.}$ $\underline{coli}$ transformants were toothpicked and subjected to in situ colony hybridization. Thirty two clones out of the 1500 colonies showed positive hybridization signals. Plasmids were isolated from eight clones selected randomly out of the 32 clones. Restriction analysis proved all of the isolated plasmids were identical harboring the 2.3 kb chromosomal fragment of $\underline{L.}$ $\underline{casei}$ .
The $\underline{E.}$ $\underline{coli}$ transformants harboring the recombinant plasmids, however, did not show LDH activity. This may due to 1) damage of LDH gene by StuI, 2) the inactive promoter of LDH gene of $\underline{L.}$ $\underline{casei}$ in $\underline{E.}$ $\underline{coli}$, or to 3) a wrong probe. To elucidate these possibilities, the nucleotide sequences of the inserted $\underline{L.}$ $\underline{casei}$ chromosomal fragments were analyzed.
The nucleotide sequence analysis revealed that all of the 17 bases of the probe matched well with the bases of the chromosomal DNA except one base "T" mismatched with "C". This C-T mismatch might have picked chromosomal DNA fragments other than the LDH gene. Since the probe was constructed correctly, more trials to hybridize the $\underline{L.}$ $\underline{casei}$ chromosomal DNA fragments with this probe would be worthwhile.
$\underline{Lactobacillus}$ $\underline{casei}$ 의 젖산 탈수소 효소 유전자의 분리를 위하여 17개의 염기로 구성된 합성 probe 혼합체를 이용하였다. 이 혼합체는 $\underline{L.}$ $\underline{casei}$ 가 생산하는 젖산 탈수소 효소의 아미노산 배열로 부터 예측되는 가능한 모든 codon 을 만족하도록 16 가지의 probe 로 구성되어 있다.
$\underline{L.}$ $\underline{casei}$ 의 염색체를 StuI으로 절단하여 Southern hybridization 한 결과 2.3 kilobase (kb) 의 염색체 조각에 probe 가 hybridize 되었다. 따라서 StuI 에 의해 절단되어 2.3 kb의 크기를 갖는 DNA조각들을 분리하여 pUC9 벡터에 ligation 한 후 $\underline{E.}$ $\underline{coli}$ JM83 을 형질전환 하였다. 이들 전환주들 중 젖산 탈수소 효소 유전자를 갖는 클론을 선별하기 위하여 in situ colony hybridization 을 수행하였다. 그러나 여기서 선별된 클론은 젖산 탈수소 효소 활성을 보이지 않았으므로 이 클론이 갖고 있는 $\underline{L.}$ $\underline{casei}$ 염색체 조각의 염기배열을 부분적으로 조사 하였다. 이 염기배열 분석으로 부터, 선별된 클론은 젖산 탈수소 효소 유전자를 갖지 않았으며 이 클론이 선별된 원인은 probe 의 3' 말단 염기인 T가 C와 짝지워져 나타난 오류 때문임을 알았다. 그러므로 제 2의 probe를 합성하여 각각의 실험과정에서 두개의 probe 를 동시에 사용함으로써 이러한 문제를 해결할 수 있을 것이다.
