Aspergillus nidulans 에서 nsdD 유전자의 cDNA 클로닝

한갑훈,한유정,한동민 원광대학교 생명공학연구소 1995 생명공학연구소보 Vol.3 No.1

In Aspergillus nidulans, Never in Sexual Development(nsdD) gene is required for the early stage of sexual development. A region of the A. nidulans genomic DNA carrying the nsdD gene which complements NsdD19 mutation was isolated by transformation of NsdD19 mutant with a cosmid library. The size of the subcloned gene was about 4kb-long and the plasmid containing the 4kb DNA fragment, pNSD19-SB4, was cloned. 2.5kb and 2. 1kb cDNAs of nsdD gene were obtained from the sexual-development specific cDNA library by plaque hybridization and cloned into pBluescriptⅡ KS+.

Aspergillus nidulans 에서 npgA 유전자의 분리

김상현,한갑훈,한동민 원광대학교 생명공학연구소 1995 생명공학연구소보 Vol.3 No.1

A gene that complements the null pigment mutation(npgA1) was isolated from Aspergillus nidulans. The mutant developed colorless conidiophores, conidia and ascospore and never secreted any diffusible pigment into the medium. A. nidulans carrying trpC802 and npgA1 was transformed with genomic DNA library constructed in a cosmid, pKBY2. Out of 2,800 trp^(+) transformants, two npg^(+) clones were selected and transforming DNA was isolated through in vitro packaging. The isolated cosmid, designated as pNPG, was further subcloned through co-transformation with pKBY2. The npgA^(+) gene was included in the 3.0Kb HincⅡ fragment.

Aspegillus nidulans 에서 정단성장이상 돌연변이주의 세포벽 구조 분석

정윤신,한갑훈,한동민 원광대학교 생명공학연구소 1997 생명공학연구소보 Vol.5 No.1

To investigate the effects of the cell wall on the pigmentation and branching in Aspergillus nidulans, the chemical composition and ultrastructure of cell wall in wild type (FGSC4), null pigmentation mutant (NPG, WX17) and suppressor of the null pigmentation mutation (SU-NPG, SU602) have been examined. The rate of protoplast formation of NPG and SU-NPG was faster than that of wild type, suggesting that cell wall structure of NPG and SU-NPG should be modified. Through the chemical analysis of cell wall it was found that the colorless phenotype of null pigmentation mutant was due to the lack of melanin in the hyphal wall. The amounts of alkali-soluble fraction, in which major component is thought to be glucan, in the cell wall extract of NPG and SU-NPG were less than that of wild type. The lack of melanin and glucan in the mutant hyphal wall might be the reason why the cell wall of mutants was more readily hydrolyzed than that of wild type when treated with the cell wall lytic enzyme Novozyme 234. Scanning electron micrographs (SEM) revealed that the most outer layer of conidia wall peeled off in NPG and SU-NPG on the 6th day from the complete of conidiation. And also SEM showed that hyphal growth and branching were developed not very well in NPG and SU-NPG. Transmission electron micrographs (TEM) showed that the plasma membrane was crenulated in wild type conidia, but that of NPG and SU-NPG was not. TEM also represented that the hyphal wall in SU-NPG was thicker tha/#n that of wild type and NPG. Also, tips were round-shaped in SU-NPG, but not in NPG and wild type. These results from electron microscope indicated that the ultrastructure of cell wall of mutants was modified.

Aspergillus nidulans에서 형질전환률을 증가시키는 DNA fragment에 관한 연구

김진희,한갑훈,한동민 圓光大學校 基礎自然科學硏究所 1996 基礎科學硏究誌 Vol.14 No.2

pNPG는 염색체 1번의 동원체 왼편에 위치하는 npgA1을 complementation할 수 있는 genomic DNA를 포함하는 이며, Aspergillus nidulans를 pNPG로 형질전환시켰을 때 형질전환율이 현저히 증가하였다. 이 때 불완전형질전환체가 형성되지 않았으며 Trp^+형질전환체 모두가 Npg^+임이 확인되었다. pNPG를 제한효소 PstⅠ으로 절단하여 얻어진 10.4kb 절편내에 형질전환율을 증가시키는 DNA서열이 존재함을 확인하였고 pILJ16 의 PstⅠ size에 재조합하여 pKJH10.4라 명명하였다. pKJH10.4로 A.nidulans를 형질전환시킨 결과 pILJ16 단독으로 형질전환했을 때보다 형질전환율이 200배 가량 증가함이 확인되었으며, 형질전환율을 증가시키는 최소한의 DNA절편은 EcoRⅠ―HaeⅢ 4.9kb 절편이었다. pNPG,a library plasmid containing genomic DNA complementing npgA1 which located on the left arm of linkage groupⅠ,transformed Aspergillus nidulans at high frequency. No abortive transformants were observed and the Trp^+ transformants were all Npg^+. The 10.4kb PstⅠ fragment was cloned into pILJ16, which increased the transformation efficiency of pILJ16 by more than 200folds. The full activity of enhanced transformation was retained on the 4.9kb EcoRⅠ―HaeⅢfragment. The DNA segment was similar to AMA1 rather than ANS1 in function and designated as AMA2.

Minireviews : Molecular Genetics of Emericella nidulans Sexual Development

Kap Hoon Han The Korean Society of Mycology 2009 Mycobiology Vol.37 No.3

The nsdD gene encodes a putative GATA type transcription factor neccessary for sexual development of Aspergillus nidulans

Han, Kap-Hoon,Han, Kyu-Yong,Han, Dong-Min 圓光大學校 基礎自然科學硏究所 2001 基礎科學硏究誌 Vol.19 No.1

The ability to reproduce both by sexually and asexually is one is of the characteristics of the homothalic ascomycetes, Aspergillus nidulans. Unlike the other Aspergillus species, A. nidulans undergoes compete sexual development and it seems to be regulated by internal and external stimuli. To begin to understand the sexual reproduction of A. nidulans we previously isolated and characterized several NSD(never in sexual development) mutants that failed to produce any sexual reproductive organs and identified four complementation groups, nsdA, nsdB, nsdC, and nsdD. The nsdD19-complementing DNA fragment has been isolated and localized within a SmaI-BamHI 4 kb fragment(pNSD19_SB4)that retained full complementing. The nucleotide sequencing revealed that the nsdD gene had an open reading frame(ORF)OF 1,565 bp initiated by AUG and interrupted by three introns of 74, 49, and 56 bp in size, respectively. The predicted NadD protein consists of 461 amino acid residues and is rich in proline and serine. peptide sequence comparison shows that NadD has the conserved amino acid sequence of type IVb C-X2-C-X18-C-X2-C zinc finger domain typically found in the GATA type transcription factors.

Regulation of nsdD Expression in Aspergillus nidulans

Han, Kap-Hoon,Han, Kyu-Yong,Kim, Min-Su,Lee, Dong-Beom,Kim, Jong-Hak,Chae, Suhn-Kee,Chae, Keon-Sang,Han, Dong-Min The Microbiological Society of Korea 2003 The journal of microbiology Vol.41 No.3

The nsdD gene has been predicted to encode a GATA type transcription factor with the type IVb zinc finger DNA binding domain functions in activating sexual development of A. nidulans. In several allelic mutants of nsdD producing truncated NsdD polypeptides lacking the C-terminal zinc finger, the transcription level of nsdD gene was greatly increased. Also in an over-expressed mutant, the transcription under its own promoter was reduced. These results suggest that the expression of nsdD is negatively autoregulated. When the nsdD gene was over-expressed, cleistothecia were formed in excess amounts even in the presence of 0.6 M KC1 that inhibited sexual development of the wild type. Northern blot analysis revealed that the expression of nsdD was repressed by 0.6 M KC1. These results strongly suggest that the inhibition of sexual development by salts was carried out via the nsdD involved regulatory network.

The conserved and divergent roles of carbonic anhydrases in the filamentous fungi <i>Aspergillus fumigatus</i> and <i>Aspergillus nidulans</i>

Han, Kap-Hoon,Chun, Yoon-Hee,de Castro Pimentel Figueiredo, Bá,rbara,Soriani, Frederico Marianetti,Savoldi, Marcela,Almeida, Agostinho,Rodrigues, Fernando,Cairns, Charlie Timothy,Bignell, Elaine Blackwell Publishing Ltd 2010 Molecular microbiology Vol.75 No.6

<P>Summary</P><P>Carbon dioxide (CO<SUB>2</SUB>) and its hydration product bicarbonate (HCO<SUB>3</SUB><SUP>-</SUP>) are essential molecules in various physiological processes of all living organisms. The reversible interconversion between CO<SUB>2</SUB> and HCO<SUB>3</SUB><SUP>-</SUP> is in equilibrium. This reaction is slow without catalyst, but can be rapidly facilitated by Zn<SUP>2+</SUP>-metalloenzymes named carbonic anhydrases (CAs). To gain an insight into the function of multiple clades of fungal CA, we chose to investigate the filamentous fungi <I>Aspergillus fumigatus</I> and <I>A. nidulans</I>. We identified four and two CAs in <I>A. fumigatus</I> and <I>A. nidulans</I>, respectively, named <I>cafA-D</I> and <I>canA-B</I>. The <I>cafA</I> and <I>cafB</I> genes are constitutively, strongly expressed whereas <I>cafC</I> and <I>cafD</I> genes are weakly expressed but CO<SUB>2</SUB>-inducible. Heterologous expression of the <I>A. fumigatus cafB</I>, and <I>A. nidulans canA</I> and <I>canB</I> genes completely rescued the high CO<SUB>2</SUB>-requiring phenotype of a <I>Saccharomyces cerevisiae</I>&Dgr;<I>nce103</I> mutant. Only the &Dgr;<I>cafA</I>&Dgr;<I>cafB</I> and &Dgr;<I>canB</I> deletion mutants were unable to grow at 0.033% CO<SUB>2</SUB>, of which growth defects can be restored by high CO<SUB>2</SUB>. Defects in the CAs can affect <I>Aspergilli</I> conidiation. Furthermore, <I>A. fumigatus</I>&Dgr;<I>cafA</I>, &Dgr;<I>cafB</I>, &Dgr;<I>cafC</I>, &Dgr;<I>cafD</I> and &Dgr;<I>cafA</I>&Dgr;<I>cafB</I> mutant strains are fully virulent in a low-dose murine infection.</P>

Environmental factors affecting development of Aspergillus nidulans

Han, Kap-Hoon,Lee, Dong-Beom,Kim, Jong-Hak,Kim, Min-Su,Han, Kyu-Yong,Kim, Won-Shin,Park, Young-Soon,Kim, Heui-Baik,Han, Dong-Min The Microbiological Society of Korea 2003 The journal of microbiology Vol.41 No.1

Aspergillus nidulans, a homothalic ascomycete, has a complete sexual reproductive cycle as well as an asexual one. Both sexual and asexual development are known to be genetically programmed, but are also strongly affected by environmental factors including nutrients, light, temperature and osmolarity. We have examined these factors to define favored conditions for fruiting body (cleistothecium) formation. In general, fruiting body formation was enhanced where carbon and nitrogen sources were sufficient. Limitation of C-source caused predominant asexual development while inhibiting sexual development. When higher concentrations of glucose were supplied, more cleistothecia were formed. Other carbon sources including lactose, galactose and glycerol made the fungus develop cleistothecia very well, whereas acetate caused asexual sporulation only. Organic nitrogen sources like casein hydrolysate and glycine, and an increase in nitrate or ammonium concentration also enhanced sexual development. In addition to nutrient effects, low levels of aerobic respiration, caused either by platesealing or treatment with various chemicals, favored sexual development. Carbon limitation, light exposure and a high concentration of salts promoted asexual development preferentially, suggesting that stress conditions may drive the cell to develop asexual sporulation while comfortable and wellnourished growth conditions favored sexual development.

골반골 악성 골 종양에서 저온 열처리 후 자가골 이식술을 이용한 치료 결과

김갑중(Kap-Jung Kim),김한수(Han-Soo Kim),강현귀(Hyun-Guy Kang),노윤환(Yoon-Whan Roh),오주한(Joo Han Oh),이상훈(Sang Hoon Lee),한일규(Il Kyu Han) 대한정형외과학회 2006 대한정형외과학회지 Vol.41 No.3

목적: 골반골의 악성 골 종양 환자에서 저온 열처리를 실시한 자가골을 이용한 재건술 후 종양학적 결과와 수술 후 기능을 평가하고자 하였다. 대상 및 방법: 1996년 1월부터 2004년 2월까지 골반골의 악성 골 종양 및 전이성 종양으로 수술한 환자 중 1년 이상 추시가 가능하였던 11예를 대상으로 하였다. 남자가 6예, 여자가 5예였으며 평균 나이는 41세였다. 이 중 원발성이 9예(골육종 5예, 연골육종 4예), 전이성이 2예(갑상선암 1예, 간암 1예)였고, 평균 추시 기간은 30개월이었다. Enneking의 골반골 절제 분류상 Type Ⅰ/Ⅱ이 3예, Type Ⅱ가 2예, Type Ⅱ/Ⅲ이 6예였다. 수술 방법은 광범위 절제, 저온 열처리 후 자가골 이식, 고관절 치환술 및 내고정을 시행하였다. 최종 추시 시 종양학적 기능 평가는 ISOLS에서 제창한 기능적 분류 척도를 이용하였다. 결과: 최종 추시 시 ISOLS 기능 점수는 61.2%이었다. 이 중 통증 지수와 정서적 만족도는 높은 점수를 보인 반면 기능, 보조 기구, 보행 능력 및 보행 양상은 상대적으로 낮은 점수를 보였다. 수술 후 평균 6개월에 골유합이 되었다. 국소 재발이 1예에서 발생하였고, 다른 합병증으로는 감염 2예, 인공 관절의 탈구 1예 등이 있었다. 결론: 골반골의 절제로 인하여 광범위한 골 결손이 생긴 경우 저온 열처리를 이용한 자가골 이식술은 단기 추시상 비교적 만족할만한 종양학적, 기능적 결과를 보였다. 동종골 이식술이 어려운 점을 감안할 때 이 방법은 골반골 재건 방법의 하나로 이용할 수 있으며 장기간의 추시가 필요할 것으로 생각된다. Purpose: This study evaluated the oncological and functional results of a surgical treatment for malignant pelvic bone tumors using a low-heat-treated autologous bone graft. Materials and Methods: Eleven patients with malignant pelvic bone tumors who were followed-up for more than one year were enrolled in this study. There were six males and five females. The mean age was forty-one years and the mean follow-up period was thirty months. Nine patients had primary bone tumors and two patients had metastatic tumors with various histological origins. A surgical resection was carried out according to the anatomic location (Type Ⅰ/Ⅱ 3 cases, Type Ⅱ 2 cases, Type Ⅱ/Ⅲ 6 cases). The surgical methods used were a wide resection, a low-heat-treated autologous bone graft, total hip arthroplasty and rigid internal fixation. The ISOLS score was used to determine the oncological outcome. Results: The mean ISOLS score was 61.2% at the final follow-up. The index of pain and emotional acceptance showed high scores, but functional ability, support, walking ability and gait showed relatively low scores. Bone union was achieved at a mean post-operative 6 months. The post-operative complications were one case of a local recurrence, two cases of infection and one case of a dislocation of the total hip arthroplasty. Conclusion: A wide resection and reconstruction with a low-heat-treated autologous bone graft in malignant pelvic bone tumors were satisfactory oncologically as well as functionally in the brief period. However, a longer follow-up and an examination of more cases will be needed.