وأيد هذا العمل من خلال منحة جوزيف هيلي من جامعة ماساتشوستس في بوسطن (LSH) وR15 GM86805 من المعاهد الوطنية للصحة (LSH). ويدعم SMP جزئيا على زمالة سانوفي-جنزايم في جامعة ماساتشوستس في بوسطن.

1. الاستعداد للتبوغ ملاحظة: وسائل الإعلام وصفها في هذا البروتوكول تتم باستخدام وصفات وطرق معيارية 13،17 يعطي الجدول 1 صياغة ل1 لتر من وسائل الإعلام المختلفة المستخدمة في هذا البروتوكول. <table border="1" cellpadding="0" cellspacing="0" height="180" width="469" style=";text-align:right;direction:rtl"><colgroup><col /><col /><col span="3" /><col /><col span="2" /></colgroup><tbody><tr height="15"><td height="15" style="height:20px;"></td><td style="width: 55px;"> Bacto ببتون </td><td style="width: 50px;"> خلاصة الخميرة </td><td style="width: 50px;"> Bacto آجار </td><td style="width: 60px;"> سكر العنب </td><td style="width: 70px;"> البوتاسيوم خلات </td><td style="width: 60px;"> الغليسيرول </td><td style="width: 60px;"> ده 2 O </td></tr><tr height="15"><td height="15" style="height:20px;"> لوحات YPG </td><td> 20 ز </td><td> 10 ز </td><td> 20 ز </td><td> - </td><td> - </td><td> 30 مل </td><td> 970 مل </td></tr><tr height="15"><td height="15" style="height:20px;"> لوحات YPD </td><td> 20 ز </td><td> 10 ز </td><td> 20 ز </td><td> 20 ز </td><td> - </td><td> - </td><td> 1000 مل </td></tr><tr height="15"><td height="15" style="height:20px;"> YPD السائل </td><td> 20 ز </td><td> 10 ز </td><td> - </td><td> 20 ز </td><td> - </td><td> - </td><td> 1000 مل </td></tr><tr height="15"><td height="15" style="height:20px;"> YPA السائل </td><td> 20 ز </td><td> 10 ز </td><td> - </td><td> - </td><td> 20 ز </td><td> - </td><td> 1000 مل </td></tr><tr height="15"><td style="height: 20px; width: 80px;"> وسائل الإعلام تبوغ </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 10 ز </td><td> - </td><td> 1000 مل </td></tr></tbody></table> يتم إعطاء صيغ وسائل الإعلام مبالغ ل1 لتر من وسائل الإعلام: الجدول 1. تفاصيل حول وسائل الإعلامالمكونات يمكن العثور عليها في جدول المواد. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إذا جميع خلايا Sporulating سوف يكون التركيب الوراثي نفسه، اتبع هذا الإجراء: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ذوبان الخميرة سلالة لاستخدامها في الصعود إلى (YPGlycerol) لوحة YPG. تنمو الخميرة ل12-16 ساعة عند 30 درجة مئوية. الأهم من ذلك، تستعد خلايا SK1 أيضا إلى sporulate، لذلك سلالات SK1 لا ينبغي أن تترك وقتا طويلا على لوحات YPG، أو أنها قد sporulate على لوحة. ملاحظة: يفضل سلالة إذابة حديثا نمت على YPG، والاستفادة من الجلسرين كمصدر الكربون يتطلب الفسفرة التأكسدية، وبالتالي يختار لالخلايا التي تحتوي الحمض النووي وظيفية. خلايا SK1 لديهم ميل لفقدان الحمض النووي وظيفية وتصبح خادمات. وهناك حاجة الحمض النووي وظيفية للتبوغ. </li><li style=";text-align:right;direction:rtl"> الخميرة متتالية من لوحة YPG الصعود إلى (YPDextrose) لوحة YPD لالمستعمرات واحد. تنمو الخميرة على لوحة YPD 24-28 ساعة عند 30 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> بعد حوالي 36-48 ساعة من النمو على YPD وسائل الاعلام الصلبة، وبدء 25 مل ثقافة YPD باستخدام مستعمرة واحدةمن لوحة YPD. تنمو هذه الثقافة حتى الخلايا حول OD 600 = 4،0-7،0. تنفيذ هذه الخطوة عند 30 درجة مئوية خلال الليل في حاضنة تهتز، تهتز في 220 دورة في الدقيقة. </li><li style=";text-align:right;direction:rtl"> من ثقافة YPD، استخدم مبلغ مناسب من الخلايا لبدء 1000 مل من YPA (YPAcetate) الثقافة التي هي OD 600 = 0.1 لكل حاجة 96 لوحة جيدا في القسم 2. تنمو الخميرة في 30 ° C هز حاضنة (اهتزاز في 220 دورة في الدقيقة) حتى ثقافة OD 600 ما بين 1،3-1،5. ملاحظة: تطعيم 100 مل من ثقافة YPA لكل لوحة multiwell 96 ليتم عرضه في القسم 2. على سبيل المثال، إذا فحص أربعة 96 لوحات multiwell، ستكون هناك حاجة إلى 400 مل من ثقافة YPA. بدءا من حجم إضافي 10٪ في الثقافة YPA غير الضرورية لحماية ضد فقدان حجم التبخر. عادة، سوف يستغرق ما بين 12-16 ساعة للخلايا للوصول الى OD المناسب 600. مقدار الوقت قد تختلف اعتمادا على التركيب الوراثي لسلالة الخميرة. YPA هو وسائل الإعلام presporulation. Pregrowthفي خلات يساعد لحث على التعبير عن الجينات (مثل IME1) اللازمة لكفاءة ومتزامن تبوغ 6. </li><li style=";text-align:right;direction:rtl"> تدور في 1800 x ج في أعلى الجدول أجهزة الطرد المركزي لمدة 5 دقائق لتكوير الخلايا المزروعة في YPA. غسل الخلايا 1X مع 0.5 حجم الماء منزوع الأيونات تعقيمها العقيمة. تدور مرة أخرى، وتجاهل يغسل. Resuspend الخلايا في وسائل الإعلام حجم تبوغ 1X. الاستمرار في القسم 2. </li></ol></li><li style=";text-align:right;direction:rtl"> في حالة استخدام الخلايا للتبوغ التي هي من المورثات المختلفة، (أي، تحولت مع البلازميدات مختلفة أو تحتوي على الطفرات المختلفة، التي تم الحصول عليها عادة وتخزينها على شكل أسهم المجمدة)، وخلايا مجموعة من التراكيب المختلفة في شكل multiwell 96. ثم اتبع هذا الإجراء: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام معقم عبة Frogger 96 الشق لنقل الخلايا من الأسهم المجمدة المذابة في 96 لوحة multiwell (ق) على YPG وسائل الاعلام الصلبة. تنمو الخميرة بين عشية وضحاها في 30 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> استخدام معقم عبة Frogger 96 الشق، ونقل الخلايا من YPG وسائل الاعلام الصلبة على YPD وسائل الاعلام الصلبة أو انتقائيةوسائل الاعلام الصلبة (أي الحد الأدنى من وسائل الإعلام الاصطناعية تفتقر الأحماض الأمينية، وإذا احتوت الخلايا البلازميدات التي تتطلب الاختيار). تنمو الخميرة عند 30 درجة مئوية حتى يتم تشكيل مستعمرات لكل سلالة، عادة 1 يوم YPD و2 أيام إذا وسائل الإعلام الانتقائي أمر ضروري. </li><li style=";text-align:right;direction:rtl"> قسامة 1.2 مل من YPD السائل وسائل الإعلام أو وسائل الإعلام السائلة انتقائية في كل بئر من 96 العميقة 2 مل لوحة multiwell. نقل الخلايا من وسائل الاعلام الصلبة إلى السائلة وسائل الإعلام باستخدام عبة Frogger، وتغطي بغطاء بلاستيكي في مكانها بواسطة الشريط، وتنمو بين عشية وضحاها في 30 درجة مئوية شاكر، والهز في 220 دورة في الدقيقة. ملاحظة: للحد من التلوث، وتغطية لوحات باستخدام الأغطية البلاستيكية من لوحة بتري مستطيلة. عقد الأغطية في مكان باستخدام قطعة من الشريط، لذلك يقتصر ذلك دوران الهواء الحد الأدنى. </li><li style=";text-align:right;direction:rtl"> لوحات أجهزة الطرد المركزي في 1800 x ج لمدة 5 دقائق، وذلك باستخدام دلو الطرد المركزي يتأرجح مع محولات لاستيعاب 96 لوحات جيدة. تخلصي من وسائل الإعلام من الخلايا. إضافة 500 ميكرولتر YPA إلى كل بئر و resuspend الخلايا مكعبات في YPA. غطاء، يغطيمع غطاء من البلاستيك الذي عقد في مكان من الشريط. </li><li style=";text-align:right;direction:rtl"> تنمو الخلايا لمدة 15 ساعة على 30 درجة مئوية في حاضنة تهتز، تهتز في 220 دورة في الدقيقة. الاستمرار في القسم 2. </li></ol></li></ol> 2. خلايا Sporulating في تنسيق Multiwell 96 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة أي واحد 3 مم معقمة حبة الزجاج الصلبة أو معقمة 5 مم × 2 مم شريط مغناطيسي في كل بئر من 96 لوحة جيدا مع 1.3 مل الآبار. (انظر ممثل النتائج أدناه للحصول على مناقشة حول ما إذا كان استخدام حبة أو بقضيب). </li><li style=";text-align:right;direction:rtl"> وإذا كان كل الخلايا ليتم عرضه من نفس النمط الجيني، انتقل إلى 2.2.1. إذا كانت الخلايا ليتم عرضه هي من المورثات مختلفة، انتقل إلى 2.2.2. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أخذ خلايا من الخطوة 1.1.5 وقسامة 500 ميكرولتر من الخلايا بالإضافة إلى وسائل الإعلام في كل بئر من لوحة multiwell 96. تغطية مع غطاء من البلاستيك الذي عقد في مكان من الشريط. المضي قدما إلى 2.3. </li><li style=";text-align:right;direction:rtl"> خلايا تدور التي هي في YPA من 1.2.4 في 1800 x ج لمدة 5 دقائق، وذلك باستخدام دلو الطرد المركزي يتأرجح مع محولات لاستيعاب 96 لوحات جيدة. صب لوسائل الإعلام و من الخلايا. إضافة 500 ميكرولتر من وسائل الإعلام تبوغ إلى كل بئر و resuspend الخلايا مكعبات في وسائل الإعلام تبوغ. تغطية مع غطاء من البلاستيك الذي عقد في مكان من الشريط. المضي قدما إلى 2.3. </li></ol></li><li style=";text-align:right;direction:rtl"> في حالة استخدام حبة الزجاج، وعينات من مكان في حاضنة تهتز عند 30 درجة مئوية ويهز في 220 دورة في الدقيقة لتبوغ. في حالة استخدام شريط مغناطيسي، وعينات من مكان على طبق من ضجة داخل 30 درجة مئوية الحاضنة للتبوغ. ملاحظة: سرعة ضجة الدقيق للقضبان تحريك مغناطيسي ليست مهمة جدا، ما دامت جميع الحانات تتحرك بنشاط وتهوية الثقافة. </li><li style=";text-align:right;direction:rtl"> لمراقبة التقدم من خلال تبوغ، سحب عينات من ثقافة sporulating عن التصور، إضافة 6 ميكرولتر من الخلايا من الثقافات sporulating إلى 24 ميكرولتر من وسائل الإعلام تبوغ في لوحة 96 ساترة جيدا (1: 5 تمييع). ترك الخلايا لتسوية لمدة 5 دقائق قبل فحص الخلايا باستخدام مجهر مقلوب. ملاحظة: في خلايا SK1 نوع البرية، والجراثيم الناضجة المدمجة شكل من 36 ساعات بعد نقلها إلى وسائل الإعلام تبوغ. تقدم من خلال تبوغ يمكن رصدها بواسطة المجهر باستخدام علامات الفلورسنت، مثل علامة النووية (أي Htb2-mCherry 18) لفحص الميتوزي أو علامة غشاء prospore (أي G20 19) لدراسة بوغ التشكل. بدلا من ذلك، وتشكيل جراثيم سهل الانكسار ويمكن رؤيته باستخدام المجهر Nomarski مدينة دبي للإنترنت بدءا حوالي 12 ساعة بعد نقل إلى وسائل الإعلام تبوغ. </li></ol>

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The authors have no competing financial interests.

هنا نقدم بروتوكول للsporulating SK1 الخميرة في شكل multiwell 96. التهوية هي المفتاح للتبوغ كفاءة، الأمر الذي يتطلب استخدام إما بقضيب أو حبة الزجاج في كل بئر. عندما يتم sporulated الخلايا في لوحة multiwell 96 في حاضنة تهتز دون أي حبة أو بقضيب، الخلايا لا sporulate بكفاءة. وينظر فقط زيادة صغيرة في ?...

وقد درس تبوغ في الخميرة في مهدها لتقديم رؤى في جوانب كثيرة من الأحياء، بما في ذلك السيطرة على الفصل كروموسوم أثناء الانقسام الاختزالي 1، آليات إعادة التركيب الجيني 2، والسيطرة على التنمية من خلال خلية يشير 3، ومراقبة التغذية التنمية 4، والنسخي تنظيم التنمية 5، والنظر في تشكيل بوغ 6. يتضمن تشكيل بوغ حدث انقسام الخلايا رواية تنطوي على تشكيل المقصورات غشاء جديدة داخل الخلية الأم تليها ترسب جدار بوغ واقية 6. هذه الدراسات أن دراسة الخلايا sporulating في كثير من الأحيان الاستفادة من sporulating بسرعة الخميرة سلالة SK1، والتي يمكن أن تخضع لعملية تكاثر في حوالي 24 ساعة بطريقة فعالة نسبيا 7،8. وعلى الرغم من وصفت تحسين ظروف تبوغ لفي مهدها الخميرة 13/9، هذه التجربةالصورة فحص تبوغ على وسائل الاعلام الصلبة أو في جو من الثقافات السائل على نطاق وحين ينفذ تبوغ من استخدام أنابيب ثقافة أو قوارير. نحن هنا تصف طريقة لsporulating الخميرة في شكل لوحة multiwell 96. نجد أن لهذا الأسلوب، تهوية أمر بالغ الأهمية للتبوغ متزامن وكفاءة، وقد ابتكرت تقنيات لضمان تبوغ كافية لصغيرة الحجم شكل multiwell. Sporulating في شكل 96 لوحة multiwell يسمح للخلايا ليتم عرضه باستخدام تقنيات عالية الإنتاجية والكواشف الأمثل لشكل لوحة multiwell، مثل هذا الفحص لالمكثفات نسخة عالية باستخدام مكتبة مزينة بالبلاط 14-16.

<table><tbody><tr><td>Nunc 1.3 ml DeepWell Plates</td><td>ThermoScientific</td><td>260251</td><td>Used for sporulation</td></tr><tr><td>Nunc 2.0 ml DeepWell plates</td><td>ThermoScientific</td><td>278743</td><td>Used for presporulation growth, step 1.2.3</td></tr><tr><td>3 mm glass bead</td><td>Fisher</td><td>11-312A</td><td>Used for sporulation</td></tr><tr><td>5 mm x 2 mm stir bar, pack of 12</td><td>Fisher</td><td>14-511-82</td><td>Used for sporulation</td></tr><tr><td>96 well frogger</td><td>V&amp;amp;P Scientific</td><td>VP407</td><td>needed for step 1.2</td></tr><tr><td>library copier</td><td>V&amp;amp;P Scientific</td><td>VP381</td><td>needed for step 1.2; to be used with the frogger</td></tr><tr><td>rectangular Petri dish</td><td>ThermoScientific</td><td>264728</td><td>needed for step 1.2</td></tr><tr><td>Bacto Peptone</td><td>BD</td><td>211677</td><td>needed for media</td></tr><tr><td>Yeast Extract</td><td>BD</td><td>212750</td><td>needed for media</td></tr><tr><td>Bacto Agar</td><td>BD</td><td>212750</td><td>needed for media</td></tr><tr><td>Dextrose</td><td>Fisher</td><td>D16-3</td><td>needed for media</td></tr><tr><td>Potassium Acetate</td><td>Fisher</td><td>P171-500</td><td>needed for media</td></tr><tr><td>Glycerol</td><td>Fisher</td><td>G33-500</td><td>needed for media</td></tr><tr><td>Black 96 well glass bottom plate</td><td>MatTek</td><td>PBK96G-1.5.5-F</td><td>needed for step 2.4</td></tr></tbody></table>

efficient sporulation of saccharomyces cerevisiae in a 96 multiwell format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

<html> لتقييم هذا البروتوكول، وتمت مقارنة الكفاءة تبوغ تم الحصول عليها من sporulating الخلايا في لوحات multiwell (كما هو موضح أعلاه) إلى خلايا sporulated استخدام كميات أكبر في قوارير (الجدول 2). وقال إن استخدام لوحات multiwell لا يحقق كفاءة عالية ينظر عندما sporulating ?...

Watch this Scientific Journal Video about Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format at JoVE.com

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

تبوغ الفعال لل<em&gt; خميرة الخباز</em&gt; في تنسيق Multiwell 96

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

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Biology

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

10.0K Views.  University of Massachusetts Boston. Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

Video: Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

تشريح السكيراء الجعوية زقاق

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

أسلوب الإنتاجية العالية عالميا لدراسة مورفولوجية عضية في البيرة س.

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

تحدد فحوصات ACT1-CUP1 الحساسيات الخاصة بالركيزة للمتحولات spliceosomal في الخميرة الناشئة

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

Genetics

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

الأيض الكمي من Saccharomyces Cerevisiae باستخدام الكروماتوغرافيا السائلة إلى جانب قياس الطيف الكتلي الترادفي

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Biochemistry

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

إعداد و Cryo-FIB micromachining من Saccharomyces cerevisiae للتصوير المقطعي كريو إلكترون

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

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.

تحليل لميكروأري السكيراء الجعوية</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.

الجينوم شاشات التنافسية للمكتبات الخميرة Barcoded

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

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

تدفق الخلوي على أساس تنقية<em&gt; S. خميرة</em&gt; بيضات ملقحة

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

توجه طريقة تطور في<em&gt; خميرة الخباز</em&gt;: المسخ مكتبة الخلق والفرز

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The fermentable carbon concentration mainly governs how the yeast cells generate ATP; thus, the variation in fermentable carbohydrate levels drives the energetic metabolism of S. cerevisiae. This paper describes a high-throughput method based on exponential yeast growth to estimate the effects of concentration changes and nature of the carbon source on respiratory and fermentative metabolism. The growth of S. cerevisiae is measured in a microplate or shaken conical flask by determining the optical density (OD) at 600 nm. Then, a growth curve is built by plotting OD versus time, which allows identification and selection of the exponential phase, and is fitted with the exponential growth equation to obtain kinetic parameters. Low specific growth rates with higher doubling times generally represent a respiratory growth. Conversely, higher specific growth rates with lower doubling times indicate fermentative growth. Threshold values of doubling time and specific growth rate are estimated using well-known respiratory or fermentative conditions, such as non-fermentable carbon sources or higher concentrations of fermentable sugars. This is obtained for each specific strain. Finally, the calculated kinetic parameters are compared with the threshold values to establish whether the yeast shows fermentative and/or respiratory growth. The advantage of this method is its relative simplicity for understanding the effects of a substance/compound on fermentative or respiratory metabolism. It is important to highlight that growth is an intricate and complex biological process; therefore, preliminary data from this method must be corroborated by the quantification of oxygen consumption and accumulation of fermentation byproducts. Thereby, this technique can be used as a preliminary screening of compounds/substances that may disturb or enhance fermentative or respiratory metabolism.

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Here we present a protocol to estimate the respiratory and fermentative metabolism by fitting the exponential growth of Saccharomyces cerevisiae to the exponential growth equation. Calculation of the kinetic parameters allows for the screening of influences of substances/compounds on fermentation or mitochondrial respiration.

Saccharomyces cerevisiae حركية النمو الهائل في الثقافة دفعة تحليل الأيض معفن والجهاز التنفسي

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The ...

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