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.

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>

Minireviews : Molecular Genetics of Emericella nidulans Sexual Development

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

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.

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.

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.

단보 : 고농도 CO2노출에 의한 Aspergillus nidulans의 유성생식 촉진효과

한갑훈 ( Kap Hoon Han ),양영석 ( Yeong Seok Yang ),김종화 ( Jong Hwa Kim ) 한국균학회 2013 韓國菌學會誌 Vol.41 No.3

진균의 분화과정은 다양한 환경요인에 의하여 영향을 받는다. 모델 사상성 진균인 Aspergillus nidulans의 경우 빛이 존재하거나 높은 염 농도, 비 발효성 당에 의하여 무성분화가 촉진되며 반대로 빛이 없거나 발효성 당이 풍부할 때, 그리고 저산소 조건일 경우 유성분화를 촉진하게 된다. 또한 veA나 nsdD와 같은 유성생식 양성조절유전자들도 유성분화를 유도하는 것으로 알려져 있다. 본 연구에서는 A. nidulans에서 CO2의 농도와 분화패턴의 관계를 알아보고자 하였다. 정상적인 조건에서 veA+야생형 균주은 유성생식과 무성생식이 균형을 이룬 상태의 분화를 진행하게 된다. 그러나 5% 정도의 높은 CO2조건에서 배양하였을 경우 무성분화는 일어나지 않고 유성생식으로만 분화과정이 일어나게 된다. 뿐만 아니라 이러한 분화 양상은 veA와 nsdD에 의존적으로 일어나게 된다. 고농도의 CO2 조건이라 할지라도 veA-혹은 nsdD- 돌연변이 균주에서는 유성분화는 일어나지 않고 무성분화만이 일어나는 것을 관찰할 수 있었는데, 이는 CO2 농도가 높아져도 유성생식에 있어서 이들 유전자의 기능이 필요하다는 것을 시사한다. 또한 5% CO2조건은 인간 병원성 진균에게 있어서 사람의 신체 내에 살아남기 위하여 적응하여야 하는 대기 조건으로, 이러한 A nidulans의 CO2농도에 따른 분화양상의 변화는 A. fumigatus와 같은 인간 병원균의 생리, 분화적 변화에 대한 비교분석에 사용될 수 있다. Fungal development is largely affected by many environmental factors. In a model filamentous fungus Aspergillus nidulans, asexual development is promoted by exposure of light, presence of salt and non-fermentable sugars. In other hand, sexual development is largely induced by absence of light, fermentable sugars and hypoxic condition. Also, some important genes including veA and nsdD play positive roles in activating sexual development. Here, we reported that the effect of high concentration of CO2 on developmental decision in A. nidulans. When wild-type veA + strain was cultured in normal condition, sexual and asexual development occurred in balanced manner. However, high concentration of CO2 (~5%) strongly activated sexual development and inhibited asexual development. Furthermore, this CO2 effect was controlled by the veA(-) or nsdD(-) gene. High CO2 culture of veA or nsdD mutant didn`t activate sexual development, suggesting that the activation of sexual development induced by high CO2 cannot overcome the genetic requirement of sexual development such as veA(-) or nsdD(-). Since 5% CO2 is an important condition for human pathogenic fungi for surviving and adapting in human body, this developmental pattern of A. nidulans affected by CO2 concentration may provide interesting clues for comparative study with human fungal pathogens including Aspergillus fumigatus.