• Journal of Microbiology
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• 제33권2호
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• pp.142-145
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• 1995

The pumb gene encoding a basic amino acid transport protein in Neurospora crassa could be cloned by using a mutant strain defective in pmb gene as a host strain, using a negative selection on the media containing amino acid analogue canavanine. To select positive transformants of the genes for cloning, an auxotrophic marker (his-2) was added to a pmb mutant strain by mating ; a triple mutant (pmn : pmb : his-2) was constructued by crossing a strain defective in basic amino acid transport system (# 1683-bat um 535 "A") to a double mutant strain defective in neutral amino acid transport and histidine production (mitrol : his-2 "a"). Crossing was performed on synthetic crossing (SC) media containing histidine. The pmn : pmb and pmn :pmb : his-2 strains were selected among the progeny colonies from crosses on plates containing 5- .mu.g/ml para-fluoro-phenylalanine (PFPA), 200 .mu.g/ml canavanine, and 500 .mu.g/ml histidine. The selected colonies were cultured on minimal media with or without histidine for discarding pmn : pmb strain, because the pmn : pmb : his -2 strain grows only on histidine containing media. The pmn :pmb : his-2 strain selected can be used as a host strain for the cloning of the pmb and the pmn genes from a Neurospora genomic library by means of positive selections.

• 미생물학회지
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• 제28권1호
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• pp.35-40
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• 1990

Thiostrepton 내성 유전자(tsr)를 포함하는 multi-copy 재조합 플라스미드 pJY7J2의 제한효소 절단지도를 작성하였다. pJY, 712는 Streptomyces에서 넓은 host range를 나타내었으며 cloning 목적에 사용할 수 있는 단일 BgtIl 제한효소 인식부위를 갖고 있었다. 플라스미드 pJY 712는 lethal zygosis(Ltz+) 현상을 보였다. pJY 712의 혁질전환빈도는 S. lividans에서 $5.0\times 10^{4}$ TFU였다. pJY 712의 Bell 제한효소 인식부위에 tyrosmase 유전자(mel)를 삽입하여 플라스미드 PJY713을 제조하였다. met 유전자를 포함한 재조합 플라스미드 pJY 714는 pJY 713의 일부분(1.9kb BgllI-BelI 단편)을 제거하여 제고하였다.

• 한국균학회지
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• 제16권4호
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• pp.247-252
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• 1988

야생형(野生型)인 만가닥버섯(Lyophyllum ulmarium ASI 8007)의 원형질체(原形質體)로부터 분리(分離)한 염색체(染色體)를 영양요구주(營養要求株)인 잔나비걸상버섯(Ganoderma applanatum ASI 7-18; cys met)의 원형질체내(原形質體內)에 $PEG+CaCl_2$로 섭입(攝入)하였다. 형질전환주(形質轉煥株)는 microtransgenome과 macrotransgenome type으로 전자(前者)는 느리고 불안정성(不安定性)인 균사생장(菌絲生長), 후자(後者)는 아주 빠른 안정성(安定性)인 균사생장(菌絲生長)이 있으며, 균사(菌絲)가 양친(兩親)보다 굵었으며 GCM+benomyl에서 균총분리(菌叢分離)가 일어났다. 이들을 전기영동으로 esterase 동위효소(同位酵素) 패턴을 조사한 결과 양친에 비하여 위치가 다르게 나타났다.

• Journal of Photoscience
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• 제1권1호
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• pp.69-73
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• 1994

Sporangiophores (spphs) of Phycomyces blakesleeanus are positively phototropic to unilateral visible (blue) light over a range of fluence rates 10$^{-9}$ to 1 W/m$^2$. The maximal bending angle is always 70-75$\circ$ from the vertical. Many mutants with abnormal phototropism have been isolated. Complementation tests revealed that the genetic grouping is completely consistent with the phenotypic classification scheme, based on sensory responses other than those to light. The spph of the piloboloid mutant, the growth zone of which gradually ceases elongation but expands spherically, and the $\beta$-carotene-overproducing mutant show negative phototropism, in contrast to the wild type spph. We hypothesized that the phototropic orientation of spph is determined by the ratio of the maximal light fluenee rate at the proximal side to that at the distal side of the spph. Based on this hypothesis, we found that the maximal bending angle was larger in thin spphs than in thick ones, and larger in spphs containing smaller amount of $\beta$-carotene than in carotene-rich spphs. In addition to our hypothesis, gravitropic experiments revealed that the maximal bending angle of the wild type spph results from a balance among positive phototropism, negative gravitropism, and the optical properties of the spph. For further advancement of this study, we developed a mutant with a high proportion of uninucleate spores, and designed an efficient microinjection method for obtaining transformants.

• 한국식물병리학회지
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• 제10권3호
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• pp.235-239
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• 1994

Viral RNA was extracted from purified Chinese cabbage strain of turnip mosaic virus (TuMV-Ca) from infected leaves of turnip. Polyadenylated genomic viral RNA was recovered by oligo (dT) cellulose column chromatography and used as a template for the synthesis of complementary DNA (cDNA). Recombinant plasmids contained cDNA ranged from about 900 bp to 2, 450 bp were synthesized. Among the selected 41 transformants, pTUCA31 and pTUCA35 had over 2 Kbp cDNA insert. Restriction endonuclease patterns of the clones examined were very similar among them. Clones pTUCA23 and pTUCA31 were overlapped with pTUA35. The longest clone pTUCA35, encoding 3'-end, showed that it contained two sites for EcoRI, and one site for BamHI, ClaI, HincII, SacI and XbaI, respectively. The restriction mapping indicated that the clone pTUCA35 contained partial nuclear inclusion body gene, complete coding region of the coat protein and 3' untranslated region of TuMV-Ca genomic RNA.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.529.2-529
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• 1986

pSp-Si (1.6kbp) was originally found in pediococcus halophilus to be a cryptic multicopy-plasmid. Hoping that the plasmid can also replicate in Bacillus subtilis, protoplast transformation of strain 207-25 (recE) was performed using pSP-Sl onto which was added the marker of tmrB8 (on 4.9 kbp EcoRI fragment ) or tmrB+ (on 0.9 kbp xbaI fragment) gene. Though the tmrB8 gene can expres tunicamycin-resistance at the single copy state, and the tmrB+ gene exerts the resistance only at the multicopy state, we could not confirm the replication of pSP-Sl (tmrB8) or pSP-Sl(tmrB+) in B. subtilis. During the experiment, however, we unexpectedly found that the circularized 0.9 kbp xgaI fragment (tmrB+) itself, which had no replication origin, could transform strain 207-25 to tunicamycin-resistant by protoplast transformation. Southern hybridization analyses with tmrB+ and other probes revealed the integration of the fragment at a single copy state into a position other than the homologous tmrB gene. This recE independent integration of another tmrB+ gene into the chromosome may contribute to the tunicamycinresistance in the transformants.

• 한국미생물·생명공학회지
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• 제23권6호
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• pp.652-658
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• 1995

We attempted simultaneous expression of genes coding for endoglucanase and $\beta $-glucosidase from Pseudomonas sp. by using a synthetic two-cistron svstem in Escherichia coli and Saccharomyces cerevisiae. Two-cistron system, 5'--tac promoter-endoglucanase gene--$\beta $-glucosidase gene-- 3', 5'-tac promoter--$\beta $-glucosidase gene--endoglucanase gene--3' and 5'-tac promoter--endoglucanase gene--SD sequence--$\beta $-glucosidase gene--3, were constructed, and expressed in E. coli and S. cerevisiae. The E. coli and S. cerevisiae contained two-cistron system produced simultaneously endoglucanase and $\beta $-glucosidase. The recombinant genes contained the bacterial signal peptide sequence produced low level of endoglucanase and $\beta $-glucosidase in S. cerevisiae transformants: Approximately above 44% of two enzymes was localized in the intracellular fraction. The production of endoglucanase and $\beta $-glucosidase in veast was not repressed in the presence of glucose or cellobiose. The veast strain contained recombinant DNA with two genes hydrolyzed carboxvmethyl cellulose, and these endoglucanase and $\beta $-glucosidase degraded CMC synergistically to glucose, cellobiose and oligosaccharide. This result suggests the possibility of the direct bioconversion of cellulose to ethanol by the recombinant yeast.

• 한국미생물·생명공학회지
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• 제19권6호
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• pp.568-574
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• 1991

효모에서 대랑의 물질생산계를 구축하기 위하여 먼저 여러 종류의 베터의 이용이 가능한 다양한 영양요구성 marker를 지니며 형질전환율이 향상된 효모숙주를 선별 개량하였다. 벡터의 제작에 사용되는 프로모터로는, 효모의 여러 유전자 중에서 그 활성이 매우 높은 해당계의 효소 GAP-DH의 구조 유전자 GAP를 이용하기로 하여, 효모염색체 DNA중에서 GAP 프로모터를 분리하여 이용하기 쉽게 변형하였다. 분리된 GAT promoter의 기능을 검토하기 위하여, reporter로 APase의 구조유전자 PHO5'를 이용하여 세포내의 copy수가 상이한 발현 벡터를 제작하여 GAP 프로모터에 의한 APase의 활성 및 전사산물을 측정한 결과, 정상적인 전사가 이루어 졌으며, 효소활성도 높게 나타났으며, 벡터의 copy수에 의한 효소활성의 차이도 감지되었다.

• 한국미생물·생명공학회지
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• 제18권6호
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• pp.560-565
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• 1990

Trichocderma koningii의 영양요구성 돌연변이주인 CUT 121로부터 얻은 원형질체를 야생형인 ATCC 26113의 핵과 혼합하여 PEG 용액을 처리한 결과, 독립 영양형의 형질전환체가 30 이상의 빈도로 생성되었다. 이 독립영양형 군체로부터 얻은 분리체 중의 하나는 xylanase의 활성이 야생형보다 3배 가량 증진되었으며, 다른 세포의 다당류 분해효소는 야생형과 유사한 수준을 나타내었다. 분리체의 DNA 함량, 인위적인 불리양상 및 동위효소 양상 등을 조사 분석한 결과, 독립영양형의 형질전환체는 실험에 사용된 형질전환체로 판명되었다. 이상의 결과로 Trichoderma속 균류의 균주개량법으로, 핵전이법이 통상의 원형질체 융합법보다 효율적인 것으로 판명되었다.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.835-842
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• 2014

Haloperoxidase (HPO, E.C.1.11.1.7) is a metal-containing enzyme oxidizing halonium species, which can be used in the synthesis of halogenated organic compounds, for instance in the production of antimicrobial agents, cosmetics, etc., in the presence of halides and $H_2O_2$. To isolate and evaluate a novel non-metal HPO using a culture-independent method, a cassette PCR library was constructed from marine seawater in Japan. We first isolated a novel HPO gene from Pseudomonas putida ATCC11172 by PCR for constructing the chimeric HPO library (HPO11172). HPO11172 showed each single open-reading frame of 828 base pairs coding for 276 amino acids, respectively, and showed 87% similarity with P. putida IF-3 sequences. Approximately 600 transformants screened for chimeric genes between P. putida ATCC11173 and HPO central fragments were able to identify 113 active clones. Among them, we finally isolated 20 novel HPO genes. Sequence analyses of the obtained 20 clones showed higher homology genes with P. putida or Sinorhizobium or Streptomyces strains. Although the HPO A9 clone showed the lowest homology with HPO11172, clones in group B, including CS19, showed a relatively higher homology of 80%, with 70% identy. E. coli cells expressing these HPO chimeric genes were able to successfully bioconvert chlorodimedone with KBr or KCl as substrate.