Name: rapid identification of chemical genetic interactions in saccharomyces cerevisiae
Uploaded: 2015-04-05T14:00:00.000+00:00
Duration: 12 min 13 s
Description: Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

CJN 실험실에서 연구 NSERC, 캐나다 암 협회 연구소 (CCSRI)와 캐나다 유방암 재단 (BC-유콘 지점)에서 운영 보조금 지원됩니다.

참고 :이 절차의 일반적인 작업은 그림 2에 설명되어 있습니다. 성장 억제 용량 1. 결정 <ol><li> 효모 하룻밤 문화의 준비 참고 :이 프로토콜에 사용되는 효모 추출물 - 펩톤 - 덱스 트로 오스 성장 미디어 (YEPD)는 표준 조리법 (13)이다. <ol><li> BY4741 (마타 HIS3 Δ1 leu2Δ0 met15Δ0 ura3Δ0) YEPD 한천 접시에 세포 밖으로 행진 30 ° C 또는 볼 식민지 형성 할 때까지 48 시간 동안 배양한다. </li><li> 하나의 식민지로 무균 배양 용기에 YEPD 액체 매체의 5 mL를 접종하여 하룻밤 문화를 준비합니다. 연속 회전이나 진탕 30 ° C에서 밤새 품어. 문화는 일반적으로 아침 (~ 2 × 10 8 세포 / ㎖)에 의해 포화에 도달 할 것입니다. </li></ol></li><li> 고체 한천 매체 준비 다양한 화학 용량을 포함 <ol><li> 여러 주식을 준비화학 물질은 적당한 용매 100X 원하는 최종 농도에서 시험한다. 참고 : 유효 농도는 화학 물질에 따라 달라집니다, 그리고 경험적으로 결정해야합니다. 높은 mM의 낮은 μM에서, 즉, 그것은 처음에 폭 넓은 최종 농도 범위를 테스트하는 것이 좋습니다. </li><li> 화학 물질의 농도에서 각각 여러 멸균 배양 관에 분취 용융 YEPD 한천 3 ㎖를 시험한다. 55 ° C의 물을 욕조에 장소 응고에서 유지합니다. </li><li> 테스트 할 화학 물질의 농도를 각각 들어 12 웰 플레이트에 중복 우물의 각 쌍에 용융 미디어, 소용돌이에 섞어 2 ~ 3 초, 피펫 1 ml에 100 배 화합물의 30 μl를 추가합니다. 만 제어 차량을 포함해야합니다. 플레이트는 실온에서 하룻밤 설정할 수 있습니다. 여분의 미디어를 폐기하십시오. </li></ol></li><li> 확산 도금 세포 <ol><li> 1 단계로 하룻밤 성장 포화 효모 문화의 2 μL와 YEPD 액체 매체 10 ㎖를 접종.1.2. </li><li> 잘 희석 주당 효모 문화의 25 μL는, 멸균 유리 구슬 또는 막대를 사용하여 균일하게 확산하고 판은 불꽃에서 건조 할 수 있도록 접시입니다. 48 시간 동안 30 ° C에서 접시를 품어. </li><li> 접시에 효모의 성장 (식민지 모두 총 개수 및 크기)을 평가합니다. 화면을 수행하는보다 10 % -15 %의 성장을 저해하지 않는 화합물의 치사량 농도를 선택한다. </li></ol></li></ol> 2. 체계적인 화학 유전자 화면 <ol><li> 소스 플레이트의 삭제 돌연변이 배열의 유지 보수 (DMA) 및 준비 <ol><li> 글리세롤 주식에서 삭제 돌연변이 어레이를 구성에 대한 자세한 설명은 Baryshnikova 등. (14)을 참조하십시오. 삭제 돌연변이가 판 당 384 식민지의 밀도로 배열되고 나면, DMA는 4 ° C에서 몇 달 동안 저장할 수 있습니다. 각각의 콜로니로 성장하는 것을 방지하기 위해 필요한만큼의 G418 200 ㎍ / ml를 함유 YEPD 한천 복제다른. 주 : 복수 직렬 복제가 느린 성장 균주의 손실을 방지하고 억제 돌연변이 출현을 최소화하기 위해 피해야한다. 이 haploids으로 컬렉션을 유지하는 경우에 특히 관련이있다. </li><li> 로봇 시스템의 배열 미생물을 사용하여 모든 복제 도금 단계를 수행합니다. 또한, 수동 피닝 도구를 사용하여 식민지를 배열 조작 할 수 있습니다. 참고 : 효모 삭제 컬렉션 여러 상용 소스에서 haploids 및 diploids로 사용할 수 있으며 일반적으로 96 웰 플레이트에 글리세롤 주식으로 제공됩니다. </li></ol></li><li> 삭제 돌연변이의 배열을 응축 <ol><li> G418 200 ㎍ / ㎖의를 포함 YEPD 한천 미디어 250 ml의를 준비합니다. G418의 유효 농도는 다를 수 있으며, 각각의 많은 경험적으로 테스트해야합니다. </li><li> 접시 당 G418 200 ㎍ / ㎖의를 포함 YEPD 한천의 50 ㎖를 주입하여 다섯 YEPD 한천 플레이트를 준비합니다. 배열을 응축하기 전에이 일일를 수행하고 판을 실온에서 냉각 할 수 있도록erature 하룻밤 다음 평평한 표면에. 참고 : 4 판은 1536 - 변형 밀도에서 수집해야합니다, 다섯 번째 판은 추가로 부어있다. </li><li> 접시 당 384 식민지의 밀도로 돌연변이 삭제 컬렉션을 포함하는 16 개의 배양 접시를 제거하고 실온 (1 ~의 시간)에 올 수 있습니다. </li><li> 섬세한 작업을 닦아 사용하여 각 판의 덮개를 형성하고 응축을 닦으십시오. 물방울이 발생할 수 있습니다 그렇게하지 ​​않으면 배열과 배열 된 돌연변이 체의 교차 오염에 증착된다. </li><li> 미생물 배열 달아 로봇을 사용하여, 판 당 1,536 식민지 (4 페트리 접시)에 접시 당 384 콜로니 (16 페트리 접시)의 밀도에서 DMA를 응축. 플레이트 (1) 핀에서 1 차 식민지가 응축 된 배열의 1 열 1 행하기 때문에이 작업을 수행 판 (2)의 1 차 식민지는 1 열 2, 2, 1 열을 행하기 판 (3)의 1 차 식민지를 행 및합니다 판 (4)의 1 차 식민지는 2 행합니다2 열. </li><li> 하룻밤 30 ° C에서 통합 된 배열을 품어. </li><li> 소스 판에서 식민지의 균일 한 전송을 보장하기 위해 배열을 검사합니다. DMA가 제대로 (그림 4A)를 응축 된 보장하기 위해 가이드 역할을 의도적으로 배열에 포함 여러 빈 위치,있을 것이다. 주 :이 시험 화합물을 함유하는 매체 상 복제본 도금 소스 판 것이다. 단일 소스 복수 pinnings 판에 사용될 수있다. 또한 많은 로봇마다 전송되는 효모의 비슷한 번호를 확인합니다 상쇄 기능을해야합니다. </li></ol></li><li> 복제 플레이트 삭제 돌연변이 어레이 <ol><li> 제어 700 ㎖ (차량 만 해당) 및 실험 매체 (화학 물질 함유) 700 ml의를 준비합니다. 이 세중 분석 (조건 당 12 판, 플러스 두 엑스트라)을 수행하기위한 충분하다. </li><li> 시험 물질 또는 플레이트 당 컨트롤을 포함 YEPD 한천의 50 ㎖를 붓고. 플레이트 t 허용오 다음 평평한 표면에 하룻밤 실온에서 냉각. </li><li> 레플리카 플레이트에 로봇을 사용하여, 실험적으로 결정된 농도로 화학 물질을 함유하는 접시의 3 세트뿐만 아니라, 차량 제어를 함유하는 플레이트 상에 세 세트 DMA 접종. 24 ~ 48 시간 동안 실온에서 접시를 품어. </li></ol></li></ol> 3. 이미징 플레이트 및 데이터 분석 <ol><li> 인큐베이터에서 접시를 제거하고 실온 (1 ~의 시간) 이전 영상에 올 수 있습니다. 이 판을 이미징하는 동안 결로 현상을 방지 할 수 있습니다. </li><li> 뚜껑을 제거하고 플랫 베드 스캐너에 얼굴을 아래로 배치하여 24 및 48 시간에서 이미지 플레이트. 적어도 300 dpi의 해상도로 이미지를 캡처합니다. 다르게는, 이미지를 캡처하는 디지털 카메라를 사용한다. 이렇게하면, 장소 플레이트는 어두운 배경에 닫 얼굴과 이미지에 뚜껑을 제거합니다. 주 : 판의 파일 형식 및 위치가 사용 정량 프로그램에 의존 할 것이다. </li&gt; <li> 정량 Balony 15 SGAtools (16), 및 ScreenMill (17) 등 여러 오픈 소스 프로그램 중 하나를 사용하여 배열을 식민지 크기 비교를 수행합니다. </li></ol> 화면의 4. 검증 참고 :이 단계에서 몇 가지 효모 삭제 돌연변이 관심있는 화학 물질 등의 과민 점수 것입니다. 이러한 화학 물질이 유전 적 상호 작용은 다음 두 가지 방법으로 확인해야합니다. 먼저 민감한 균주의 신원은 PCR에 의해 확인되어야한다. 둘째, 화학 감도는 독립적으로 득점해야합니다. 아래에 설명하는 변형 과민, 효모 생물학에서 일반적인 기술을 검증하기 위해 안보 분석을 수행하기위한 간단한 프로토콜입니다. <ol><li> 킬 밖으로 G418 200 ㎍ / ㎖의를 포함 YEPD 한천에 효모 삭제 컬렉션 (과민) 돌연변이를 원했다. 콜로니를 형성 할 때까지 30 ° C에서 품어. 프라이머에 따라 함께 진단 PCR에 의해 변형의 유전자형 확인제조업체의 프로토콜입니다. </li><li> 각 변형에 대한 YEPD 액 5 mL를 접종하고 회전이나 진탕 30 ° C에서 밤새 품어. </li><li> OD 600을 측정하고 멸균 dH보다 2 O 600 = 1 중독 각 균주를 희석 이들은 지금 정규화 된 효모 문화입니다. </li><li> 96- 웰 플레이트의 웰 A1로 정규화 야생형 효모 (BY4741) 100 ㎕ 배양 디스펜스. 우물 B1-H1에 최대 7 개의 추가 균주의 100 μl를 분배. </li><li> A6-H6를 통해 우물 A2-H2 멸균 dH보다 2 O의 90 μl를 분배하는 멀티 채널 피펫을 사용합니다. 마지막으로, 표준화 된 효모 배양 1:10 희석 시리즈를 준비합니다. 직렬 멀티 채널 피펫으로 열 2 열 1의 10 μl를 피펫 팅에 의해 시작, 6 희석이 이루어질 때까지 96 웰 플레이트에 걸쳐 계속 (즉, 10 0-10 -5). </li><li> 멀티 채널을 사용하여 그리드에 희석 효모 문화의 2-5 μl를 전송화학 및 차량 컨트롤을 포함 YEPD 고체 매체에 피펫. 24 ~ 48 시간 동안 적절한 온도에서 접시를 품어. </li><li> 판을 검사하고 (BY4741 컨트롤을 기준으로) 화학 물질을 선택 돌연변이의 감도를 비교합니다. 이미지 (24) 접시와 단계 3.2에서와 같이 48 시간. 참고 : 관련 유전자 온톨로지 또는 함수 여러 민감한 균주의 동정은 스크린의 유효성을 확신 적합하지만, 유전자의 야생형 카피를 함유하는 플라스미드 과민 결실 균주의 변환은 공식적 유전자 것을 설정하는 데 필요한 관심의 삭제 (그리고 숨겨진 두 번째 사이트 돌연변이) 약물 감도가 발생합니다. </li></ol>

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The authors declare they have no competing financial interests.

화학적 상호 작용 유전자 프로파일을 생성하기위한 방법이 여기에 설명했다. 방법은 간단하다 화학의 유무에 광범위한 유전자 결실 컬렉션 각 균주의 콜로니 크기를 비교하여, 화학적 내성 모독에 필요한 모든 유전자들은 식별된다. 민감한 균주의 결과 목록의 주석 농축 분석은 화학 물질의 작용 모드에 대한 통찰력을 제공하기 위해 사용될 수있다. 이 프로토콜은 효모 신진 위해 최적화되어 있?...

유전 도구와 모델 생물 사카로 미세스 세 레비 시아에 이용 가능한 자원 일괄 유전자 네트워크 생물학적 시스템의 요구 사항을 이행하는 방법으로서 기능에 대한 새로운 통찰력을 제공 대규모 기능 유전체학 연구있게되었다. 이러한 도구의 초석은 효모 1, 2의 모든 열려있는 독서 프레임의 비 필수 유전자 결실의 완전한 세트의 공동 제작했다. 눈에 띄는 관찰 표준 실험실 조건에서 haploids로 성장하면 효모의 유전자 만 ~ 20 %가 생존에 필요한 것이 었습니다. 이것은 다른 생물학적 경로의 이용을 통해 게놈 섭동에 대하여 버퍼링 세포의 능력을 강조한다. 유전 개별적으로 가능한 있습니다 돌연변이하지만 조합에 치명적인는 연결 또는 수렴 병렬 생물학적 경로를 신호 및 생물학적 기능을 설명하는 유전자 상호 작용 네트워크를 형성한다. 조건부 TE의 발전과 함께필수 유전자의 mperature에 민감하고 hypomorphic 대립이 기술은 중요하지 않은 유전자 3,4의 연구에 국한되지 않았습니다. 이 개념은 유사한 세포 과정에 관여하는 유전자 (5)가 함께 클러스터링 방법을 설명하는 편견 유전자 상호 작용지도를 생산하는 게놈 규모로 적용되었습니다. 유전자 네트워크의 화학 교란 모방 유전자 결실 (그림 1) 6. 과민성위한 결실 균주의 고밀도 배열 대해 성장 저해 화합물을 조회하면 화학적 스트레스를 허용하는 데 필요한 유전자의리스트, 즉 화학적 상호 작용 유전자 프로파일을 식별한다. 유전 적 상호 작용과 마찬가지로, 화학 라이브러리의 대형 스크린은 액션 함께 클러스터 (7)의 모드를 갖는 화합물과 유사한 것으로 나타났다. 따라서, 화합물의 화학적 상호 작용 유전자 프로파일을 구축하여 작업의 모드는 LAR과 비교하여 추론 할 수있다GE의 규모의 합성 유전자 및 화학 유전자 상호 작용은 8,9 데이터 집합. 화합물의 점수가, 심문하고있는 큰 규모의 화학 유전 스크린, 바코드 경쟁 분석에 의해 수행되었다. 이 방법에서는, 삭제 균주의 풀링 된 컬렉션 화학 물질을 포함하는 미디어의 작은 볼륨에서 여러 세대에 한꺼번에 성장한다. 각 삭제 돌연변이가 고유 한 유전자 바코드를 품고 있기 때문에, 삭제 균주의 풀 내의 개별 돌연변이의 생존 / 성장은 마이크로 어레이 또는 높은 처리량 시퀀싱 (10)에 의해 추적됩니다. 생체 활성 화합물을 함유하는 고체 아가상에서 성장 물리적으로 배열 된 돌연변이 콜로니 크기를 모니터링하여 적응도를 추론하는 것은 화학적 상호 작용 11,12 유전자를 확인하는 효과적인 방법이다. 이 방식은 경쟁 기반 스크리닝에 비용 효율적인 대안을 제공하고, 화학 물질의 작은 라이브러리를 분석하기에 적합하다.여기에 설명 된 S.의 화학적 상호 작용 유전자의리스트를 생성하기위한 간단한 방법이다 생물학 조작 또는 인프라 분자에 의존하지 않는 cerevisiae의는. 그것은 단지 효모 삭제 수집, 로봇 또는 수동 피닝 장치 및 무료로 사용할 이미지 분석 소프트웨어가 필요합니다.

<table><tbody><tr><td>Yeast Extract</td><td>BioBasic</td><td>G0961</td><td>For YEPD liquid/solid media add to 1% final concentration (w/v)</td></tr><tr><td>Tyrptone Powder</td><td>BD Biosciences</td><td>211820</td><td>For YEPD liquid/solid media add to 2% final concentration (w/v)</td></tr><tr><td>Dextrose</td><td>Anachemia</td><td>31096-380</td><td>For YEPD liquid/solid media add to 2% final concentration (w/v) &amp;#8212; do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving.</td></tr><tr><td>Agar A</td><td>Bio Basic</td><td>FB0010</td><td>For YEPD solid media add to 2% final concentration (w/v)</td></tr><tr><td>G418</td><td>A.G. Scientific Inc.</td><td>G-1033</td><td>Prepare 1,000x stock at 200&amp;nbsp;mg/ml in dH&lt;sub&gt;2&lt;/sub&gt;O and filter sterilize.&amp;nbsp;</td></tr><tr><td>12-well plate</td><td>Greiner Bio One</td><td>655180</td><td></td></tr><tr><td>5 ml culture tubes</td><td>Evergreen Scientific</td><td>222-2376-080</td><td></td></tr><tr><td>10 cm Petri Dish</td><td>VWR</td><td>25384-302</td><td></td></tr><tr><td>ROTOR HDA</td><td>Singer Instruments&amp;nbsp;</td><td>ROT-001</td><td>high-throughput microbial array pinning robot</td></tr><tr><td>PLUSPLATE&amp;copy; Petri Dish</td><td>Singer Instruments&amp;nbsp;</td><td>PLU-001</td><td>Box of 200 dishes</td></tr><tr><td>384 Short-Pin RePad</td><td>Singer Instruments&amp;nbsp;</td><td>RP-MP-384</td><td>Box of 1,000 pads</td></tr><tr><td>1536 Short-Pin RePad</td><td>Singer Instruments&amp;nbsp;</td><td>RP-MP-1536</td><td>Box of 1,000 pads</td></tr><tr><td colspan="4">Alternative Pinning Tools:</td></tr><tr><td>Fully Automated Robtic Systems</td><td>S&amp;amp;P robotics</td><td>http://www.sprobotics.com</td><td>Several automated colony handling robitic and imagining systems available.</td></tr><tr><td>Manual Pinning Tools</td><td>V&amp;amp;P Scientific</td><td>http://www.vp-scientific.com</td><td>Handheld replication tools and accessories.&amp;nbsp;</td></tr></tbody></table>

rapid identification of chemical genetic interactions in saccharomyces cerevisiae

생리 활성 화학 물질의 작용 모드를 결정하는 학술, 제약의 넓은 범위에 관심, 산업 과학자이다. 사카로 미세스 세 레비 시아, 또는 신진 효모, 모델 진핵 생물이되는 ~ 6,000 유전자 삭제 돌연변이 hypomorphic 필수 유전자의 전체 모음 돌연변이 체는 시판되고있다. 이러한 돌연변이의 수집 체계적 화학 견딜 필요 유전자 화학적 상호 작용, 즉 유전자를 검출하는데 사용될 수있다. 이 정보는, 차례대로, 화합물의 작용 가능성 모드에보고한다. 여기에서 우리는 효모 신진 화학 - 유전자 상호 작용의 신속한 식별을위한 프로토콜을 설명합니다. 우리는 액션의 잘 정의 된 메커니즘이 화학 요법 제 5- 플루오로 우라실 (5-FU)을 사용하는 방법을 보여준다. 우리의 결과는 핵 TRAMP가 엑소 및 DNA 복구 효소 이전 m과 일관성이 5-FU의 존재하에 증식에 필요한 RNA 보여기반 바 코딩 화학 유전 적 접근과 5-FU에 악영향을 모두 RNA 및 DNA 신진 대사에 영향을 미치는 것을 지식을 icroarray. 이러한 높은 처리량 스크린 필요한 검증 프로토콜은 또한 기술된다.

이 방법의 유효성으로서 우리는 위에서 설명한 프로토콜은 다음 화학 요법 제 5- 플루오로 우라실 (5-FU)의 대표적인 화학적 상호 작용 유전자 스크린을 수행 하였다. 5-FU는 티미 신타뿐만 아니라 DNA와 RNA 대사 (18)을 방해하는 것으로 알려져있다. 5-FU의 화학적 유전 적 상호 작용은 공부 잘하고 이질 동형 접합 삭제 컬렉션 8,19를 모두 사용하여 효모 바코드 마이크로 어레이 기술에 ?...

Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

화학 유전자 상호 작용의 신속한 식별<em&gt; 사카로 미세스 세 레비 시아</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

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Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

10.3K 조회수.  University of Victoria. Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

비디오: Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

ChEC-seq와의 단백질 - DNA 상호 작용의 게놈 전체 매핑<em&gt; 사카로 마이 세스 세레 비시 애</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

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유전학

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

분열 효 모 Schizosaccharomyces pombe 에 깊은 시퀀싱 지원, 자발적인 억압 화면

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

게놈 전체 상호 Hemizygosity 분석을 통해 사카로 마이스 효모의 종 간의 열내성 차이의 유전 매핑

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiae의의 양적 proteomics와 단백질 단지의 식별

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

생물학

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

사카로마이세스 세레비시아에서 근접 결찰 및 정량적 PCR을 통한 상동 재조합 중간체의 검출

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

사카로미세스 세레비시아스 스플리케소터 서브콤플렉스의 과도성 행위자 검출을 위한 그라픽스의 활용

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

생화학

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Microarray 분석을위한 Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Barcoded 효모 게놈 라이브러리의 경쟁력 화면

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

성장 기반의 결정 및 단백질 분해에 대한 유전 요구 사항의 생화학 적 확인에<em&gt; 사카로 미세스 세 레비 시아</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Schizosaccharomyces pombe에서 다중 복제 억제 인자의 식별을위한 유전자 스크리닝

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

화학 유전자 상호 작용의 신속한 식별

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ChEC-seq와의 단백질 - DNA 상호 작용의 게놈 전체 매핑