Systematic targeted gene deletion using the gene-synthesis method in fission yeast

Nam, Miyoung,Lee, Sook-Jeong,Han, Sangjo,Kim, Dongsup,Lee, Minho,Kang, Eun-Jung,Park, Han-Oh,Lee, Ah-Reum,Lee, Sol,Kim, Cheol-Hee,Kim, Dong-Uk,Hoe, Kwang-Lae Elsevier 2014 Journal of microbiological methods Vol.106 No.-

<P><B>Abstract</B></P> <P>Genome-wide targeted gene deletion, a systematic method to study gene function by replacing target genes with deletion cassettes, using serial-PCR or block-PCR requires elaborate skill. We developed a novel gene-synthesis method to systematically prepare deletion cassettes on a 96-well basis in fission yeast. We designed the 2129-bp deletion cassette as three modules: a central 1397-bp KanMX4 selection marker module and two flanking 366-bp gene-specific artificial linker modules. The central KanMX4 module can be used in multiple deletion cassettes in combination with different sets of flanking modules. The deletion cassettes consisted of 147 oligonucleotides (93 for the central module+25 for each of the flanking modules+4 for the joints) and the oligonucleotides were designed as ~29mers using an in-house program. Oligonucleotides were synthesized on a 96-well basis and ligated into deletion cassettes without gaps by ligase chain reaction, which was followed by two rounds of nested PCR to amplify trace amounts of the ligated cassettes. After the artificial linkers were removed from the deletion cassettes, the cassettes were transformed into wild-type diploid fission yeast strain SP286. We validated the transformed colonies via check PCR and subjected them to tetrad analysis to confirm functional integrity. Using this method, we systematically deleted 563 genes in the fission yeast <I>Schizosaccharomyces pombe</I> with a >90% success rate and a point-mutation rate of ~0.4 mutations per kb. Our method can be used to create systematic gene deletions in a variety of yeasts especially when it included a bar-code system for parallel analyses.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The deletion cassette was designed as three modules consisting of ~29-mer oligos. </LI> <LI> The oligonucleotide set of the KanMX4 module was commonly used. </LI> <LI> Ligation chain reaction was optimized at 58.3°C and 40cycles. </LI> <LI> Nested PCR using artificial linkers yielded sufficient quantity of deletion cassette. </LI> <LI> The gene-synthesis method yielded a success rate of >90% gene deletion. </LI> </UL> </P>

Duplication and deletion of 21 hydroxylase gene among the normal Korean subjects and in adrenogenital syndrome patients

Jin, Dong-Kyu,Beck, Nam-Seon,Oh, Phil-Soo,Whang, Hye-Zin,Koh, Si-Whan,Kim, Jung-Sim,Oh, Myung-Ryurl Korean Society of Medical Genetics 1997 대한의학유전학회지 Vol.1 No.1

Steroid 21 hydroxylase deficiency is a major cause of congenital adrenal hyperplasia (CAH) and is caused by genetic impairment of the gene (CYP21B). In the human genome, CYP21B is located within the MHC class III region on the short arm of chromosome 6. Most of the genes in this region are highly polymorphic and crowded. Also the CYP21B gene is accompanied by its pseudogene (CYP21A) and tandemly arranged with two genes of fourth component of complement. This highly complex gene cluster in this area may predispose genetic instability of CYP21, i.e. mutations. In this study, tried to investigate the frequency of duplication and deletion of CYP21 and patterns of the genetic alterations of these genes.We also compared the genetic alteration in normal subjects with those of the CAH patients. The results showed that 15% of the normal korean population have duplication or deletion of CYP21. There was one normal subject with heterozygous deletion of CYP21B. Of the 5 CAH patients examined, 2 were found to show abnormal patterns. One was a large-scale gene conversion and the other a gene conversion associated with deletion involving both CYP21B and C4 locus II gene. Through this study, we carne to the conclusion that the duplication or even deletion of CYP21 and C4 might be quite a common event in the Korean population and these rearrangements must be regarded as polymorphisms. It could contribute to a high incidencs of CAH by providing a genetic pool of instable CYP21.

Duplication and deletion of 21 hydroxylase gene among the normal Korean subjects and adrenogenital syndrome patients

Dong Kyu Jin,Nam Seon Beck,Phil Soo Oh,Hye Zin Whang,Si Whan Koh,Jung Sim Kim,Myung Ryurl Oh 대한의학유전학회 1997 대한의학유전학회지 Vol.1 No.1

Steroid 21 hydroxylase deficiency is a major cause of congenital adrenal hyperplasia (CAH) and is caused by genetic impairment of the gene (CYP21B). In the human genome, CYP21B is located within the MHC class Ⅲ region on the short arm of chromosome 6. Most of the genes in this region are highly polymorphic and crowded. Also the CYP21B gene is accompanied by its pseudogene (CYP21A) and tandemly arranged with two genes of the fourth component of complement. A highly complex gene cluster in this area may predispose genetic instability of CYP21, i.e. mutations. In this study, we tried to investigate the frequency of duplication and deletion of CYP21 and patterns of the genetic alterations of these genes. We also compared the genetic alterations in normal subjects with those of the CAH patients. The results showed that 15% of the normal Korean population have duplication or deletion of CYP21. There was one normal subject heterozygous for the deletion of CYP21B. of the 5 CAH patients examined, 2 were found to show abnormal patterns. One was a large-scale gene conversion and the other a gene conversion associated with deletion involving both CYP21B and C4 locus Ⅱ gene. Through this study, we came to the conclusion that the duplication or even deletion of CYP21 and C4 might be quite a common event in the Korean population and these rearrangements must be regarded as polymorphisms. It could contribute to a high incidence of CAH by providing a genetic pool of instable CYP21.

Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics

Francesco Calì,Giuseppa Ruggeri,Mirella Vinci,Concetta Meli,Carla Carducci,Vincenzo Leuzzi,Simone Pozzessere,Pietro Schinocca,Alda Ragalmuto,Valeria Chiavetta,Salvatore Miccichè,Valentino Romano 생화학분자생물학회 2010 Experimental and molecular medicine Vol.42 No.2

A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH)alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation-dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8(systematic name c.353-?_912 + ?del) and exon 6(systematic name c.510-?_706 + ?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics.

Allelic Deletions of Chromosomes in Human Colorectal Cancer Development

Song, Young Tack,Kim, Seung Nam,Lee, Jai Hak,Yoo, Seung Jin,Cho, Won Il,Chang, Suk Kyun,Choo, Sang Yong CATHOLIC MEDICAL CENTER 1993 Bulletin of the Clinical Research Institute Vol.21 No.2

Two types of genetic alterations have been reported in colorectal tumors. The first type involves point mutations in ras proto-oncogenes. The second type of alterations involves detection of specific chromosomal regions. Deletions can be detected by restriction fragment length polymorphism analyisis of tumor DNA. The deleted sequences have been hypothesized to include tumor suppressor gene. The inactivation of tumor-suppressor gene by deletion of suspected locus lead to neoplatic growth. To investigate the relation between ras point mutation and allelic deletion of chromosome 17p and 18q, both p-PCR and RELP analysis using VNTR marker and synthetic oligonulectide probe tailing with digoxigenin Ⅱ-dUTP were done in 15 normal mucosa and 22 colorectal cancer mucosa. The obtained results were as follows: 1. Incidence of ras point mutations was 80% (4/5) in Dukes’ B, 82.3% (14/17) in Dukes’ C. There was increasing tendency of mutation along with stage progression, but no statistical significance was noted. 2. Incidence of ras point mutations were 45.4% (5/11) in well differentiated cancer and 77.7% (7/9) in moderately differentinted cancer. 3. Incidence of allelic diletion of 17p pYNH37 were 80% (5/11) in Dukes’ B, 64,7% (l1/17) in Dukes’ C. There was no correlation of incidences of 18q OS-4 in colorectal cancer. But incidence of 17p allelic deletion was higher than 18q allelic deletion in Dukes’ C colorectal cancer. The allelic deletions of pYNZ22 were noted in 6 cases of Dukes’ C colorectal cancer but not in Dukes’ B colorectal cancer. 4. There was no statistical difference between incidence of ras point mutation and allelic deletion in colorectal cancer. These results showed there was no statistical difference in incidence of ras point mutation & 18q allelic deletion in colorectal cancer. 17p allelic deletion occurred more frequently in advanced colorectal cancer. Genetic alteration did not have any correlation with cellular differentiation or tumor location in the biologic behavior of the colorectal cancer.

glpD and glpE 유전자의 조절영역 결손변이주기가 전사조절에 미치는 영향

정희태,정수열,최용락 東亞大學校附設遺傳工學硏究所 1996 遺傳工學硏究 Vol.- No.3

glpD와 glpE는 같은 조절 영역 하에서 121 bp 간격을 두고 역방향으로 전사되어지며, 이들 유전자 상류영역에 존재하는 하나의 CRP binding site가 두 유전자의 전사 조절에 관여함을 이미 여러 연구를 통하여 확인되어졌다. 본 연구에서는 glpD와 glpE의 조절영역이 다양한 크기로 결손된 변이주를 작성하였다. 각 변이된 promoter의 발현조절 영향을 보고자 LacZ와의 융합 plasmid를 작성하였고, β-galactosidase 활성을 측정하여 cAMP에 의한 발현조절을 검토하였다. 발현조절 특성을 확인한 결과 CRP binding site 전까지 결손이 된 변이주는 wild와 비슷한 활성이지만 CRP bin-ding site가 없어진 변이주의 경우는 promoter 활성이 크게 감소했고 cAMP에 의한 발현조절은 나타내지 않았다. 한편 CRP의 공통배열을 삽입시키면 회복이 가능하였다. glpE의 조절영역도 glpD와 마찬가지로 다양하게 결손시킨 변이주를 작성하여 promoter 조절활성을 확인한 결과 CRP binding site가 없어진 변이주의 경우는 promoter 활성이 크게 감소하였으며, 결손된 CRP 결합영역을 삽입시키니 cAMP에 의한 전사촉진이 거의 회복되어졌다. The glpD gene encoding gly-3-p dehydrogenase is essential for the aerobic growth of E. coli on glycerol or gly-3-p. The glpE gene, the function of which is unknown, is transcribed divergently with respect to glpD gene. Expression of the adjacent but divergently transcribed glpD and glpE genes is positively regulated by the cAMP-CRP complex. In this study, for a precise investigation of the functional elements in the regulatory region for transcription activation by cAMP-CRP, deletion mutation have been introducted into the regulatory region. The effect of the deletion mutant on transcriptional regulation was tested in vivo by β-galctosidase activity. Deletion mutants in the regulatory region of glpD demonstrated that the presence of the CRP-binding site resulted in an sixfold increase in promoter activity. And also deletion mutants of glpE gene demonstrated that the presence of the CRP-binding site resulted in an eightfold increase in promoter activity. Insertion of 22 bp oligomer in the deletion mutants has shown that the CRP binding site is need for maximal expression of glpD and glpE genes.

PCR-based S.pombe transformation for systematic deletion mutants and functional analysis of human gene

(Sung Hou Lee) 상명대학교 공학기술연구소 2008 공학기술연구 Vol.2008 No.0

S. pombe의 genomic sequence 염기서열의 분석결과에 따르면 유전자의 기능적 반복성이 적으며 S. pombe에는 진핵세포에서 중요한 세포의 기능과 질병에 관련된 유전자들이 많은 양 차지하고 있다고 보고되었다. 따라서 S. pombe는 약물개발을 위한 cell-based 시스템으로서 인간 유전자의 기능연구에 효율적인 방법으로 쓰일 수 있다. 본 실험에서는 S. pombe의 genome-wide systematic deletion mutant 제조를 완성하기 위한 기반을 마련하기 위하여 이미 deletion mutant가 제조 된 바 있는 두 개의 유전자인 sum1, gef3를 직접 deletion 하여 gene deleted S. pombe를 제조 해 보았다. sum1은 S. pombe에서 essential gene으로써 chromosome 1에 존재하고, gef3은 non-essential gene으로써 chromosome 2에 존재한다. 이렇듯 gene의 essentiality를 증명하기 위하여 deletion mutant를 만드는 과정 중 yeast transformation을 위한 DNA 단편의 제조를 완성 하였으며, agarose gel electrophoresis를 통하여 확인 할 수 있었다. 이와 같이 임의로 제조된 DNA 단편은 S. pombe에 transformation 시킴으로써 mutant를 만들기 위한 핵심 요소가 될 것이다.

Duchenne/Becker 근이영양증에서의 Dystrophin 유전자 분석

박수연(Su Yeon Park) 고경남(Kyung Nam Koh) 임병찬(Byung Chan Lim) 강호석(Ho Seok Kang) 이경연(Kyoung Yeon Lee) 황희(Hee Hwang) 채종희(Jong Hee Chae) 최지은(Ji Eun Choi) 김기중(Ki Joong Kim) 황용승(Yong Seun Hwang) 대한소아신경학회 2004 대한소아신경학회지 Vol.12 No.1

목적 : 국내 Duchenne/Becker 근이영증에서의 dystrophin 유전자 결실은 빈도 및 그 분포를 알아보고 유전형과 임상형의 관계를 분석하여, 국내 유전 역학 자료를 마련하고 좀 더 효과적인 유전 상담 및 예방 방법을 모색해보고자 하였다. 방법 : 1999년 1월부터 2003년 7월까지 서을대학교병원에서 상기 질환으로 진단 후 추적 관찰중인 89명의 환자를 대상으로 하였다. 임상 진단 후 근생검을 시행, dystrophin 면역조직화학 염색을 하였으며 dystrophin 유전자 결실은 혈액에서 추출한 DNA를 이용, multiplex PCR 방법을 이용하여 진단하였다. 결과 : 국내 DMD/BMD 환자의 dystrophin 유전자 결실률은 54%였고, 결실 부위의 hot spot은 exon 44-54 부위로 전체 결실 환자의 80%를 차지하였다. 결실이 확인되지 않았던 22명의 환자에서 23개의 exon을 대상으로 직접염기서열 분석을 시행한 결과 6명의 환자에서 6종류의 점돌연변이를 발견하였다. 유전형과 임상형의 관계에 대한 분석 결과 중심형 결실이 근위형에 비해, 다중 결실이 단일 결실에 비해 증상 발생 연령이 어리고 진행이 다소 빠른 경향을 보였으나 통계적으로 유의한 관련성은 찾을 수 없었다. 결론 : 국내 DMD/BMD 환자의 결실률은 54%였으며 hot spot은 exon 44-54였다. 따라서 혈액 유전자 검사로 진단할 수 없는 약 50%의 환자의 진단을 위해서는 근생검 및 dystrophin 항체 면역 조직화학 검사를 통한 진단이 시행되어야 할 것이다. 또한 세밀한 가족력 조사를 포함하여 적절한 보인자 검사를 통해 합리적인 유전 강담을 진행하고 이에 기초한 질병 발생 예방 및 교육 등이 필요할 것으로 생각된다. Purpose : Duchenne/Becker muscular dystrophy(DMD/BMD) is an X-linked recessive disorder caused by mutations of dystrophin genes. The purpose of the present study is to determine the frequency and the patterns of dystrophin gene deletions and to investigate the correlation of genotypes and phenotypes. Methods : There were included a total of 89 children(88 boys and I girl) diagnosed as DMD/BMD by immunohistochemistry and/or genetic analysis from 1999 to 2003 at Seoul National University Children´s Hospital. We analyzed the genomic DNA by multiplex PCR using a 26 dystrophin exon primer set. Direct sequencing was performed on 23 exons(in which point mutations were detected in other previous reports) in 22 patients without deletions. Phenotype and genotype relationship analysis was performed on the basis of retrospective clinical reviews. Results : The frequency of dysmorphin gene deletions was 54%(32/59), which is lower than that of European and American data. Exon deletions were detected in 59 cases and the deletion "hot spots" were exon 44-54 constituting 80% of all deletions. In 6 cases without detectable deletions, 6 point mutations(3 nonsense mutations and 3 nucleotide variants) were detected The patients whose deletions were in the central parts or the patients with multiple exon deletions tended to show earlier symptom onsets and more rapid progressions of weakness but there were no statistical significances. Conclusion : Since deletions in dystrophin genes were detected in about 50% of the patients, studies on dystrophin protein expressions using muscle biopsy samples must be done for correct diagnosis.

Intragenic deletion patterns of dystrophin gene in Duchenne and Becker muscular dystrophy patients from Algeria

Amira Cherrallah,Tarik Hamadouche,Traki Benhassine,Sonia Nouioua,Samira Makri,Malika Chaouch,Meriem Tazir 한국유전학회 2014 Genes & Genomics Vol.36 No.1

Duchenne and Becker muscular dystrophies(DMD and BMD) represent the most frequent neuromusculardiseases in humans (1/3,500–6,000 live male births),characterized by an X-linked recessive pattern of inheritanceand therefore affecting mainly male individuals. DMD and BMD are allelic disorders resulting from geneticdefects, mostly intragenic deletions, in the dystrophin gene. Using multiplex polymerase chain reaction (PCR), we haveanalyzed 170 male patients from unrelated families originatingfrom Algeria, showing that 68 % of them harboreddeletion events affecting the known 50 or 30 hot spotregions. The distal portion was predominantly involved(85 %), whereas 37 distinctive patterns of deletion wereidentified in our panel. The extent of deletion varied from 1to 32 exons, although the average number was about fourexons. The lack of seven exons (45, 46, 47, 48, 50, 51 and 52), each alone or in combination, represented about 78 %of the alterations encountered, while exon 48 was mostfrequently involved (50 %). The effect of the deletionsshowed that the reading frame rule proved mostly true,correlating with the clinical diagnosis suggested. Moreover,the c.525delT mutation in the c-sarcoglycan gene waspresent in non-deleted patients (7 %), suggesting thatclinical features can still be misleading. Finally, multiplexPCR proved to be a simple, fast and low-cost approach forthe molecular diagnosis of dystrophinopathies in Algeria,whereas our data could contribute to the creation of anational registry of DMD/BMD patients in our country,which would give them hope to an access to alreadyavailable genotype-based therapies.

Genetic Analysis of Dystrophin Gene for Affected Male and Female Carriers with Duchenne/Becker Muscular Dystrophy in Korea

이보련,남숙현,이준화,기창석,이문향,이지훈 대한의학회 2012 Journal of Korean medical science Vol.27 No.3

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked recessive disorders caused by mutation in dystrophin gene. We analyzed the results of a genetic test in 29 DMD/BMD patients, their six female relatives, and two myopathic female patients in Korea. As the methods developed, we applied different procedures for dystrophin gene analysis; initially, multiplex polymerase chain reaction was used, followed by multiplex ligation-dependent probe amplification (MLPA). Additionally, we used direct DNA sequencing for some patients who had negative results using the above methods. The overall mutation detection rate was 72.4% (21/29) in DMD/BMD patients, identifying deletions in 58.6% (17/29). Most of the deletions were confined to the central hot spot region between exons 44 and 55 (52.9%, 7/19). The percentage of deletions and duplications revealed by MLPA was 45.5% (5/11) and 27.2% (3/11), respectively. Using the MLPA method, we detected mutations confirming their carrier status in all female relatives and symptomatic female patients. In one patient in whom MLPA revealed a single exon deletion of the dystrophin gene, subsequent DNA sequencing analysis identified a novel nonsense mutation (c.4558G > T; Gln1520X). The MLPA assay is a useful quantitative method for detecting mutation in asymptomatic or symptomatic carriers as well as DMD/BMD patients.