Genes and Gene Action 2 Course Guide 2019-2020

Genes and Gene Action 2 Course Guide

2019-2020

Course Organiser: Prof.David Finnegan Dr Elizabeth Bayne

Name: ______

If you require this document in an alternative format, such as large print or a coloured background, please contact the Biology Teaching Organisation, University of Edinburgh, James Clerk Maxwell Building, King’s Buildings, Edinburgh or email Carolyn Wilson at [email protected] ASSESSMENT DEADLINES AND FEEDBACK

Assessment Available Due Feedback % of % of ICA Course mark mark Course Problem Jan 24th Feb 17th - noon Tutorial 3 12.5% 5% Lecture Quiz 1 Jan 21st - 11am Jan 28th - 11am Quiz 2 Jan 28th - 11am Feb 4th - 11am Quiz 3 Feb 4th -11am Feb 11th -11am Quiz 4 Feb 11th - 11am Feb 25th - 11am On Quiz 5 Feb 25th -11am Mar 3rd -11am submission 25% 10% Quiz 6 Mar 3rd - 11am Mar 10th -11am Quiz 7 Mar 10th -11am Mar 17th -11am Quiz 8 Mar 17th -11am Mar 24th - 11am Quiz 9 Mar 24th - 11am Mar 31st -11am PeerWise Jan 17th Day of Exam After exam 12.5% 5% Data Handling April 1 April 1 On 50% 20% Test Submission The semester 2 exam diet will run from Monday 27th April 2020 to Friday 22nd May 2020. Exam dates will be published on Monday 2nd March 2020.

Who to contact if you... If you don’t know what tutorial or practical group you are in Need timetable information then check your online timetable or Learn If you still need help, ask at BTO reception. *Complete the Group Change Request form at https://www.edweb.ed.ac.uk/timetabling- examinations/timetabling/personalised-timetables/group- change-request Have a timetable clash *Please note that due to the high number of students enrolled on the course this year it may not be possible to move you to your chosen group and you may be asked to arrange to swap groups with a fellow student. Need general GGA2 information Email the course secretary: [email protected]

No need to contact anyone. Check the Learn site as lecture Have missed a lecture notes and recordings are uploaded regularly. If possible speak to others who have attended and make sure that you understand any problems and solutions that Have missed a practical or tutorial have been discussed. Should any points of difficulty remain you should seek clarification from your tutor or practical leader Contact your Personal Tutor or Student Support Tutors. For Have health or personal problems details see ‘Special Circumstances’ section of the “Essential Information for Biological Science Students” Need academic advice about the Contact the Course Organiser: [email protected] course: Have a question about a specific Email the relevant Lecturer. lecture

Student intranet: https://www.wiki.ed.ac.uk/display/SBSUndergraduateIntranet/School+of+Biological+Scienc es+Undergraduate+Students%27+Intranet

GENES AND GENE ACTION 2019 - 2020

CONTENTS

Assessment deadlines 1 Aims of Course 3 Learning Objectives 3 Graduate Attributes 3 Timetable 5 Teaching Staff 6 Recommended Textbooks 7 Course discussion board 7 Structure of the Course 7 Synopsis of Lectures 8 Lecture “flipping” 10 Organisation of Practical and Tutorial Groups 11 Schedule of Practicals and Tutorials 12 Lab and Tutorial records 13 Assessment 14 PeerWise 14 Course Problem 14 Data Handling Test 15 Exam 15 Common Marking Scheme 17 Feedback (to you and from you) 18 FAQ 19 Tutorial 1 21 Tutorial 2 23 Tutorial 3 25 Tutorial 4 26 Synopsis of Practicals 29 Practical Schedule 31 Practical 1 32 Practical 2 36 Practical 3 46 Practical 4 55 Practical 5 59 Further Information 65

GENES AND GENE ACTION 2 2019-2020 This guide should be used in conjunction with information and learning support material available on Learn.

AIMS OF THE COURSE The course aims to provide an introduction to modern genetics to prepare students for third year courses. This is achieved through an integrated series of lectures, practicals and tutorials designed to give an appreciation of the systems and methods used for classical genetic and molecular genetic analysis.

LEARNING OUTCOMES As a result of successfully completing this course you should: 1] be able to explain how genetics is used to analyse a variety of biological phenomena. 2] be able to carry out genetic experiments and to analyse and interpret the results of these experiments. 3] be able to solve genetic problems related to the topics covered during the course 4] be able to use collaborative learning to develop your personal effectiveness.

DEVELOPMENT OF “GRADUATE ATTRIBUTES’ The University has identified a set of four clusters of skills and abilities that students should develop throughout their degree programme to strengthen your attitude towards lifelong learning and personal development, as well as future employability. The graduate attributes we hope to develop with the Genes and Gene Action 2 course are indicated below.

Research and Enquiry This course aims to increase your understanding of classic, modern and molecular genetics and also your problem solving skills. The knowledge obtained, and the development of research and technical skills, will be of benefit to you during the rest of your degree and beyond. The course will develop your research and problem solving capabilities through your work on the course problem and the data handling test, and the feedback that you will obtain from these tasks. Staff will assist you to learn and practice problem-solving skills during tutorials and practicals throughout the course.

Personal and Intellectual Autonomy You are expected to take responsibility for your own learning and to work independently to meet the challenges of the course. You are expected to explore textbooks, and occasionally research papers, not only to expand your knowledge of the topics covered in the lectures, but to broaden your understanding of areas of genetics that interest you. You are encouraged to discuss the course problem and questions in the tutorials and practicals with other students but answers that you submit must be entirely your own work. You are also responsible for making your own record of practical and tutorial exercises. These are integral part of the course and will help you when preparing for both the data handling test and the degree exam while peer marking of the course problem will develop your ability to assess the ideas of others.

Communication Through discussion and collaboration with students in practical and tutorial groups you will be able to communicate your views and ideas and to learn from your peers. Genes and Gene Action has an active Learn discussion board and you can benefit greatly from participating. You are also required, as part of the course assessment, to engage in peer marking, and to use the online PeerWise system to share and discuss course related questions. The opportunities to benefit academically from such communications are proven and considerable.

Personal Effectiveness Throughout your degree programme you will learn transferable skills that will benefit you not only across the courses you are enrolled in, but in future employment and further study. In this course, as in others, time management is an important skill that you will learn as you develop ways to organise your work and meet deadlines. Effective participation in the Peer wise exercise, for example, requires that you submit your questions reasonably early in the course in order to allow others to answer, comment and discuss your questions with you. Working effectively as a member of a group is an important transferable skill. This will be enhanced by collaborating with other students in tutorials and practicals. This will help you become aware of your skills and talents (and possibly your limitations) and to develop your own scientific style.

TIMETABLE Week Date Lectures and Quectures Practicals Tutorials Quizzes Title Lecturer # Groups Tutors Start End 14-Jan 1: Introduction – DF DF – – – – 1 15-Jan – – Practical 1 – Group 1 (am); Group 2 (pm) – – – 16-Jan 2: Recombination and Maps 1 DF – – – – 17-Jan 3: Recombination and Maps 2 DF – – – – 21-Jan 4: Recombination and Maps 3 DF – – – Quiz 1 – 2 22-Jan – – Practical 1 – Group 3 (am) 1 A to F HW, HM, AD, JG, DF, VM 23-Jan 5: Thinking about Genes DF Practical 1 – Group 4 (pm) 1 G, H & Q HW, KH, JS 24-Jan 6: Mendelian Genetic Analysis HM – – – 28-Jan 7: Chromosome Structure HM 1 S DF Quiz 2 Quiz 1 3 29-Jan – – Practical 2 – Group 1 (am); Group 2 (pm) 1 I to N HW, HM, AD, JG, DF, VM 30-Jan 8: Eukaryotic Genes and Genomes HM – 1 O, P & R HW, KH, JS 31-Jan 9: Genetic Interactions HM – – – – 04-Feb 10: DNA Replication MeK – – – – Quiz 3 Quiz 2 4 05-Feb – – Practical 2 – Group 3 (am) 2 A to F HW, HM, AD, JG, DF, VM 06-Feb 11: Mutation, Mutagenesis and Repair MeK Practical 2 – Group 4 (pm) 2 G, H & Q HW, KH, JS 07-Feb 12: Gene Expression: Transcription and Translation MeK – – – – 11-Feb 13: The Prokaryotic Gene: structure and regulation MeK – 2 S DF Quiz 4 Quiz 3 5 12-Feb – – Practical 3 – Group 1 (am); Group 2 (pm) 2 I to N HW, HM, AD, JG, DF, VM 13-Feb 14: The Genetic Code and Consequencs of Mutation MeK – 2 O, P & R HW, KH, JS 14-Feb 15: Plasmids DL – – – – 18-Feb No quiz – 19-Feb 20-Feb Flexible Learning Week 21-Feb 25-Feb 16: Bacterial Transpsons DL – – – Quiz 5 Quiz 4 6 26-Feb – – Practical 3 – Group 3 (am) 3 A to F HW, HM, AD, JG, DF, VM 27-Feb 17: Conjugation DL Practical 3 – Group 4 (pm) 3 G, H & Q HW, KH, JS 28-Feb 18: Transduction and Transformation DL – – – 03-Mar 19: Mid-semester feedback and Revision Session DF, MeK, DL HM – 3 S DF Quiz 6 Quiz 5 7 04-Mar – – Practical 4 – Group 1 (am); Group 2(pm) 3 I to N HW, HM, AD, JG, DF, VM 05-Mar 20: PCR and DNA Sequencing VM – 3 O, P & R HW, KH, JS 06-Mar 21: Recombinant DNA Technology VM – – – 10-Mar 22: Nucleic Acid Hybridization VM – – – Quiz 7 Quiz 6 8 11-Mar – – Practical 4 – Group 3 (am) 4 A to F HW, HM, AD, JG, DF, VM 12-Mar 23: Problem Solving and Data Handling Test DF Practical 4 – Group 4 (pm) 4 G, H & Q HW, KH, JS 13-Mar 24: Genetic Control of Development – the toolkit JG – – – 17-Mar 25: Mutants and Genetic Analysis of Developoment JG 4 S DF Quiz 8 Quiz 7 9 18-Mar – – Practical 5 – Group 1 (am); Group 2 (pm) 4 I to N HW, HM, AD, JG, DF, VM 19-Mar 26: From Genetic Screens to Developmental Function JG – 4 O, P & R HW, KH, JS 20-Mar 27: Segmentation and the Role of Homeotic Genes JG – – – 24-Mar 28: The Cell Cycle KH – – – Quiz 9 Quiz 8 10 25-Mar – – Practical 5 – Group 3 (am) – – 26-Mar 29: Cell Division and Cell Death KH Practical 5 – Group 4 (pm) – – 27-Mar 30: Cancer KH – – – 31-Mar – – – – – – Quiz 9 11 01-Apr – – DATA HANDLING TEST – – 02-Apr – – – – – 03-Apr – – – – – 23-Apr Revision Surgery – 14.30-15.30: JCMB 3217

Lectures : Tuesday and Friday 10.00 – 10.50am, Thursday 12.10 – 1.00pm, Swann Lecture Theatre Practicals: Wednesday 10.00am – 1.00pm, OR 2pm – 5pm, OR Thursday 2pm – 5pm, Teaching Laboratory, JCMB room 1104 but meet in Swann Lecture Theatre at the start of each session Tutorials: Tuesdays 2pm– 4pm, OR Wednesdays 10am – 12 noon, OR Thursdays 2pm – 4pm. Locations and groups are on Learn and MyTimetable Lecturers: DF=David Finnegan, DL=David Leach, MeK= Meriem El Karoui, HM=Heather McQueen, VM=Vaso Makrantoni, JG=Justin Goodrich, KH=Kevin Hardwick Tutors: AD = Anne Davidson, HW=Helen Wallace, JS=Jo Strachan, VM=Vaso Makrantoni,

Lecturers DF: David J Finnegan, Room 1.69 Roger Land Building email: [email protected] JG: Justin Goodrich, Room 1.02D Daniel Rutherford Building email: [email protected] KH: Kevin Hardwick, Room 6.25 Swann Building email: [email protected] MeK: Meriem El Karoui, Room 1.11 Roger Land Building email: [email protected] DL: David Leach, Room 1.12 Roger Land Building email; [email protected] HM: Heather McQueen, Room 1.64 Roger Land Building email: [email protected] VM: Vaso Makrantoni: Room 6.24 Swann Building email: [email protected]

Tutors (in addition to lecturers) AD: Anne Davidson email: [email protected] JS: Jo Strachan email: [email protected] HW: Helen Wallace email: [email protected]

Practical Leader Nadia Tuzi: Biology Teaching Organisation email: [email protected] Hazel Cruickshank: Biology Teaching Organisation email: [email protected]

Course Administrator Carolyn Wilson BTO, Room 2015B, James Clerk Maxwell Building Email: [email protected]

RECOMMENDED TEXTBOOKS

The primary text for the course is: “An Introduction to Genetic Analysis” 11th ed, by Griffiths, Wessler, Carroll and Doebley covers most of the material in this course. It will be useful for 3rd and 4th year courses in genetics and would be a good choice if you wanted to buy a book of your own. Earlier editions, back to about edition 7, should also be helpful.

The following texts are good sources of information for particular aspects of the course:

“Molecular Biology of the Gene” 7th ed. By Watson et al. is an excellent source for the more molecular aspects of the course.

“Human Molecular Genetics” 4th ed. By Strachan and Read is an excellent source of information on human genetics.

Each of these books can be borrowed from the library.

If you would like to kearn more about various aspects of genetics you may like to listen the Genetics Unzipped (https://geneticsunzipped.com), the podcast of the UK Genetics Society.

COURSE DISCUSSION BOARD The discussion board on Learn allows you to post questions about the course that can be answered by your fellow students or teaching staff. It has sections for questions relevant to each aspect of the course and you may post questions and answers or comments anonymously if you wish. Questions of general relevance to students that are sent to staff by email will be posted on the discussion board along with the response.

STRUCTURE OF THE COURSE Lectures and tutorials are synchronised so that you will explore data and concepts in each tutorial that are relevant to material recently given in lectures. Practical material also relates to lecture content but has a stand-alone timetable and most experiments extend over more than one practical.

The course content covers aspects of both classical and molecular genetics and prokaryotic and eukaryotic organisms considering recombination and genetic exchange, DNA replication and gene structure and expression, modern genetic techniques and how they may be applied, the genetic control of development, and finally cell biology and cancer.

LECTURES These lectures will be interactive and will include questions for you to answer during the lecture using Top Hat. Information about the use of Top Hat follows the lecture synopses

Lectures 6–9 and Lectures 20–22 will take the form of ‘Quectures’ for which preparation will be given on Learn and is ESSENTIAL. Please come prepared. More information about quectures follows the lecture synopses.

Lecture synopses

1. Introduction to Genes and Gene Action 2019-2020.

2. Recombination and Maps 1: Meiosis and genetic linkage.

3. Recombination and Maps 2: Mapping from three point crosses, recombination frequency and chiasma frequency

4. Recombination and Maps 3: Genetic maps and physical maps. Mitotic recombination.

5. Thinking About Genes” The complementation test. Intragenic complementation. Complementation and recombination within a gene.

6. Mendelian Genetic Analysis: Single gene inheritance. Autosomal or sex chromosome genes, and their phenotypes. Restriction digestion.

7. Chromosome Structure: Relating structure to function for the main features of eukaryotic chromosomes.

8. Eukaryotic Genes and Genomes: Features characteristic of eukaryotic gene organisation and expression

9. Genetic Interactions: Dominant or recessive phenotypes. Multifactorial traits and their underlying genes

10. DNA Replication: The basic mechanism of DNA replication. Replication in prokaryotes (E.coli). Replication in eukaryotes; the end-replication problem, telomeres and telomerase.

11. Mutation, Mutagenesis and Repair: Spontaneous DNA mutation and repair mechanisms. Mutagenesis, screening and selection.

12. Gene Expression: Transcription and Translation: The basic mechanisms of transcription and translation.

13. The Prokaryotic Gene: structure and regulation: Why regulate gene expression? Operons and Regulons. Negative and positive control: lac, trp and AraC examples.

14. The Genetic Code and Consequences of Mutation: Elucidation and general features of the code. Effects of DNA mutation. Intergenic and intragenic suppression of mutation.

15. Plasmids: Extrachromosomal elements, plasmid replication and transmission. Drug resistance. Transposition.

16. Bacterial Transposons: The discovery of transposable elements. The structure of bacterial transposons. The mechanism of transposition.

17. Conjugation: F and the formation of Hfr strains, mobilisation of chromosomal DNA, mapping by interrupted mating.

18. Transduction and Transformation: Mapping by recombination. Generalised transduction by bacteriophage P1. Transformation.

19. Mid-semester Feedback and Revision Session for Lecture 2 – 18

20. PCR and DNA Sequencing: Methodology and applications of Polymerase Chain Reaction (PCR). Use of quantitative PCR (qPCR). Sanger and Next Generation sequencing technologies and their role in identifying disease genes.

21. Recombinant DNA Technology: Detecting genes by hybridisation (Southern blotting, northern blotting, in situ). Screening libraries by hybridisation. Use of DNA microarrays to study the transcriptome and comparison to RNA deep sequencing.

22. Nucleic Acid Hybridisation: Making and purifying recombinant plasmids. Construction of libraries. Gibson Assembly. Applications of Recombinant DNA technology.

23. Problem Solving and the Data Handling Test

24. The Genetic Control of Development – the ‘toolkit’: Preformation vs epigenesis, the concept of the organiser. Embryonic development – how do cells become different (‘differentiate’)? Control of selective gene expression – enhancers and transcription factors. Cell interaction and signalling pathways (eg Hedgehog). Polydactyly and limbless snakes.

25. Mutants and Genetic Analysis of Development: Systematic genetic screens for identifying maternal-effect and zygotic genes. Balancer chromosomes as a tool to maintain recessive lethal mutations. Use of transposons to tag and clone developmental patterning genes.

26. From Genetic Screens to Developmental Function: Maternal effects in anterior posterior patterning of Drosophila embryo development. bicoid, the Bicoid gradient, hunchback transcription.

27. Segmentation and the Role of Homeotic Genes: Ed Lewis and the identification of homeotic genes, their role in segment identity, their conservation. Role of Ubx in insect leg and wing development. Role of Hox genes in specifying body plan in vertebrates

28. The Cell Cycle: Principles and regulatory mechanisms. cdc mutants, MPF, Cdk-cyclin complexes, ubiquitin-mediated proteolysis and the concept of checkpoint controls.

29. Cell Division and Cell Death: Mitosis, cytokinesis and apoptosis. How genetic screens in yeasts and nematodes identified molecular components of these complex cellular processes.

30. Cancer: The cell cycle “out of control”. The identification of oncogenes and tumour suppressor genes. Genome re-arrangements and aneuploidy.

All lectures in the course will be recorded and will be available on Learn following each lecture.

Top Hat To help you understand topics discussed in the course most lectures will include questions to which you can supply answers using Top Hat. This is a personal response system that allows you to use a web enabled device such a smart phones, tablets and laptops to engage actively with material presented during lectures. If you have not used it before you should find out how to get connected and to register with the system by going to https://www.ed.ac.uk/information-services/learning-technology/electronic-voting-system/students.

The Top Hat course code for Genes and Gene Action 2020 is 028888

‘Quectures’ Lectures 6–9 and Lectures 20–22 will be ‘Quectures’, a form of the ‘flipped classroom’. In this form of learning, preparation is done by students in advance of the lecture period to make room for question based interactive learning during the lecture slot. In this way students can use (rather than simply remember) the new concepts and information, to form a deeper understanding of the new material. Preparation material will be available on Learn one week in advance of each quecture and this will be assumed to have been done. This material will not be presented again during the quectures. The preparation will include a short quiz, the results of which will contribute to the 10% of course points allocated for lecture quizzes across the course. Average preparation time should be around one hour per lecture although this may vary between students. Use of Top Hat will be an essential component of the quectures.

PRACTICALS AND TUTORIALS Practicals and tutorials are an integral and essential part of this course and satisfactory attendance is required to meet the learning outcomes of the course. There is a strong positive correlation between attendance at tutorials and practicals and passing the course and you are asked to sign in at practicals and tutorials so that we can identify students who may be having difficulties. Should you be unable to attend any practical or tutorial, it is essential that you make sure that you understand all the work covered and/ or that you speak to your practical floor-leader or tutor. If you are allocated to a timeslot that you have difficulty in attending, you should consult the BTO to request a group change as soon as possible.

There will be no practicals or tutorials during the Festival of Creative Learning (17th – 23rd February)

Your knowledge of practical and tutorial material will be assessed through a data handling test on 1st April as well as during the final examination in May.

Practicals You will be allocated to one of four practical groups (1-4) as listed on Learn. Half the class will start the practicals on Wednesday 15th January, while the other half will start theirs on Wednesday 22nd or Thursday 23rd January. They will be held in the Teaching Laboratory, room 1104, in the James Clerk Maxwell Building (JCMB), King’s Buildings and each practical will be introduced by a short talk given in Swann Lecture Theatre.

Practicals on Wednesday mornings: Group 1 [start 15th January] Group 3 [start 22nd January]

Practicals on Wednesday afternoons: Group 2 [start 15th January]

Practicals on Thursday afternoons: Group 4 [start 23rd January]

The experiments are described in this course guide that you must bring to each session and you should record your experiments and results in your laboratory book. Although they do not involve handling hazardous chemicals or equipment you must wear a lab coat, which will be provided for your use, whenever you are in the practical laboratory and wear gloves and safety glasses when instructed to do so (both are provided). A COSHH risk assessment for these practicals is available for anyone who wishes to see it. You will be asked to do a limited amount of clearing up at the end of the practical and must observe standard lab hygiene (no eating or drinking) and wash your hands when finished. Get your demonstrator to explain anything you don’t understand. Remember it is the role of demonstrators to help you. They are very willing to do this but can only help with a difficulty if you tell them what it is.

Practicals are located in: Teaching Laboratory, room 1104 JCMB, 10.00am – 1.00pm OR 2.00pm – 5.00pm (Meet in Swann Theatre at the start of each practical) Practical Date Practical content 1 Jan 15th Prepare genomic DNA for PCR experiment; Set 1 Jan 22nd/ 23rd up bacterial conjugation; Analyse three-point cross data.

2 Jan 29th Set up PCRs; Set up yeast genetics experiment; 2 Feb 5th / 6th Prepare plasmid DNA; Complete bacterial conjugation.

3 Feb 12th Gel electrophoresis of PCRs; Restriction digests 3 Feb 26th/27th of plasmid DNA; Continue yeast genetics experiment; Genetics problem; -galactosidase activity (lac operon) and DNA fragment size estimation exercises.

4 Mar 4th Gel electrophoresis of plasmid DNA fragments; 4 Mar 11th/12th Conclude the yeast Genetics experiment; Set up bacteriophage T4 rII intragenic recombination; Analysis of PCR products; Observe plates from the lac operon exercise; Construct plasmid map.

5 Mar 18th Conclude the bacteriophage T4 rII intragenic 5 Mar 25th/26th recombination experiment; Construct plasmid map and analyse; Gene mapping in humans.

Tutorials You will be allocated to one of eighteen tutorial groups as listed on the course noticeboard outside the JCMB teaching laboratory and on Learn. Tutorials are held on Wednesday mornings or Thursday afternoons and alternate with the practicals, but these do not start until Week 2.

Tutorials on Tuesday afternoons: Group S [start 28th January]

Tutorials on Wednesday mornings: Groups A to F [start 22nd January] Groups I to N [start 29th January]

Tutorials on Thursday afternoons: Groups G, H & Q [start 23rd January] Groups O, P & R [start 30th January]

The tutorials are based around genetics problems related to the lectures. The questions are designed to improve both your understanding of material in the course and your ability to solve problems. The questions are given in the course guide and you should attempt them before the tutorial as you will derive most benefit from these tutorials by preparing the work beforehand and identifying the parts causing you most difficulty.

The tutorials are also an opportunity to seek help with any of the lecture material about which you are unclear, and for further discussion of topics of particular interest to you. They may vary in length but will usually last about two hours. 12

Laboratory notebooks and tutorial records Model answers will not be provided for the questions in tutorials and practical. You must keep your own written record of the results of your experiments and the answers to the tutorial questions. Before leaving the laboratory or tutorial make sure you have understood and have written answers to all questions. If you do have a problem then ask a demonstrator or tutor for help. You need not write long descriptions of practicals or tutorials and there is no need to duplicate what is in this manual, however your work should be complete. In particular your records should enable you to revise the material in preparation for the exams. The data handling test and some questions in the degree exam will be based on the practicals and tutorials.

ASSESSMENT OF THE COURSE Your achievements in the course will be assessed in two ways, through In-Course Assessments that together contribute 40% of the final mark, and an exam in May that contributes 60% of the final mark.

In-Course Assessments (ICA) The ICA comprises a peer–marked course problem (5%), Post lecture and pre-quecture quizzes (10%), PeerWise (5%), and a data handling test (20%).

Peer-marked Course Problem (5%) The problem will be made available via Learn at 12.00pm on Friday 24th January. The deadline for submitting your answer is noon on Monday 17th February. You must submit an electronic version using the dropbox on Learn. You should not submit a hard copy but should bring one to Tutorial 3 when the problem will be discussed. The usual late penalties apply and an extension to the deadline will only be given if there are special circumstances and an application is made before the deadline.

The purpose of this problem is to help you develop your problem solving skills. You will benefit from working collaboratively on this problem but the answer that you submit must be entirely in your own words and all answers will be checked for plagiarism.

The problem will be peer-marked via Learn. You will mark your own answer, and will mark and comment on the answers of two of your colleagues assigned at random. In the same way your answer will be marked and commented on by two others. Marking the answers of your colleagues will help you identify the strengths and weaknesses in your work, as will the comments that are made by the students who mark it. You must complete the peer marking by 12.00pm on Friday 20th March.

Marks will be assigned as follows: For getting at least 40%, and marking the answers assigned to you 5% For getting at least 40% but not marking the answers assigned to you 4% For getting less than 40% and marking the answers assigned to you 3% For getting less than 40% but not marking the answers assigned to you 2% For not submitting an answer and not marking the answers assigned to you 0%

Marks for the problem will be released on or before Friday 20th March

Lecture quizzes (10%) There will be nine post-lecture quizzes designed to test your understanding of the material in the lectures. These will be made available on Learn following the Tuesday lecture and you will have one week in which to answer the questions. Each quiz will contain six questions, two based on material in each of the preceding three lectures. There will be a pre-lecture quiz for each Quecture and these will contribute to your mark for this component of assessment.

PeerWise (5%) PeerWise is an online collaborative learning tool with which you will create, answer and discuss with other GGA students course-related multiple choice questions. Creation of challenging questions that explore common misconceptions and build upon your previous learning, is an ideal way to expand your knowledge and conceptual understanding of course-related material. Further information on how and why to use PeerWise is given at http://peerwise.cs.auckland.ac.nz/docs/students/. 14

You will have been enrolled on PeerWise GGA unless you joined the course late, in which case you should contact the course Administrator, Carolyn Wilson and ask her to enroll you. Before you can use PeerWise you will have to register by going to http://peerwise.cs.auckland.ac.nz/register/?ed_uk and find “Genes and Gene Action 2020” by entering the course ID 20509 and your student identifier that is your matriculation number without the “s”. You will have access to PeerWise until the day of the final exam.

Your mark for PeerWise will be based on your contributions and activity up to the day of the May exam as indicated below:

If you complete the minimum requirement and achieve a reputation score of at least 3,500 5% If you complete the minimum requirement and achieve a reputation score of 1,500 – 3,499 4% If you complete the minimum requirement and achieve a reputation score of less than 1,499 3% If you do not complete the minimum requirement 1% If you do not participate 0%

The minimum requirement is to create between 2 and 10 questions and answer at least 20 questions and comment on, and rate, 5 questions

The reputation score is calculated automatically and is based on your activity in each of the three areas (question setting, answering and commenting). Submitting questions from early in the course will help you to achieve the necessary reputation score and will help you to perform better in the course overall. Only submitting questions at the end of the course will not allow you to achieve a reputation score of more than 3,500.

Data Handling Test (20%) This test contains questions similar to those that you have experienced in the Practicals and Tutorials. It is an online test for which you will have 90 minutes if required by a learning profile. It will be held in the Hugh Robson Building computer suite on Wednesday 1st April 2020. Because of the size of the class there will be two sittings starting at 9.30 am and 11.45 am. You will be informed of the time of your own test at least a week in advance and should bring writing implements (pencil, eraser and ruler) for workings and/or graph drawing, and an approved calculator to carry out calculations. Some example question will be discussed in Lecture 24 and you will have access to a short online practise test.

The exam The exam will have three parts: Part A, 20 multiple-choice questions (MCQs) (20%); Part B, a course problem (20%); and Part C, one long answer question from a choice of six (LAQ) (20%).

The exam period will be from 27th April to 22nd May 2020 and the date and location of the exam will be published on the 2nd March. You can search for this information at http://www.scripts.sasg.ed.ac.uk/registry/examinations/index.cfm.

You should bring to the exam a pen for your written answers, a pencil, eraser and ruler for diagrams or graphs, and an approved calculator, as described in the BTO “Essential Information for Biological Science Students”, in case you need it for calculations in the problem. The long 15

answer questions will be essay-style questions and your answer should be written as logical prose that addresses the question fully using clear language. You are encouraged to use diagrams as appropriate but you should not answer in bullet points or unconnected sentences. You answer will be marked according to the common marking scheme that is included on the next page of this booklet. Further guidance is available on Learn.

PASSING THE COURSE To pass the course, you must satisfy all of the following criteria:

1. Have an overall weighted aggregate mark of at least 40% 2. Have an overall mark of at least 40% for the in-course assessments. 3. Have an overall mark of at least 40% for the exam. 4. Have demonstrated that you have met the learning outcomes of the course.

Your ability to analyse and interpret genetic data will be assessed by the course problem, the data handling test, and the problem in the exam. In some borderline cases, and at the discretion of the exam board, a narrow failure in the problem solving component(s) of one part of the course may be made good by doing well in the problem solving component(s) of the other.

If you get less than 40% for either the in-course assessments or the exam, or both, then a fail will be recorded on your transcript unless there are special circumstances associated with your failure and these are accepted by the Special Circumstances Committee. You will have an opportunity to re-sit the components of the course for which you got less than 40% either during the summer or the re-sit exam diet, You will be required to pay a re-sit fee for this.

External Examiner Prof. Graeme Ruxton, School of Biology, University of St Andrews

Class Medal A class medal will be awarded to the student, or students, with the best overall performance in the course. This will awarded during Welcome Week in September 2020.

THE COMMON MARKING SCHEME

HONOURS NON-HONOURS

Honours Class Mark % Grade Non-Honours Description 90-100 A1 1st 80-89 A2 Excellent 70-79 A3 2.1 60-69 B Very Good Performance at a level showing the potential to 2.2 50-59 C achieve at least a lower second class honours degree Pass, may not be sufficient for 3rd 40-49 D progression to an honours programme 30-39 E Marginal Fail 20-29 F Clear Fail Fail 10-19 G Bad Fail 0-9 H

DESCRIPTION OF WHAT IS EXPECTED FOR EACH GRADE Class Description First Work which is excellent in the breadth of knowledge and command of the material covered, and in its argument and analysis. Work that has shown originality and treated the evidence critically, and which brings in relevant material from a wide range of sources. The work should be well- organised and complete. Higher marks (A2 Grade, 80-89%) should be awarded for a performance meeting all, or virtually all, of these criteria. An A3 Grade (70-79%) answer would meet several of the criteria. An A1 Grade (90-100%) should only be awarded in very exceptional cases for work that is truly outstanding (top 1%). Upper Work that shows a very good and broad-based knowledge of the topic 2nd that is presented in an organised way, and is clearly and critically argued, and focused on the set question. Lower Work that is good and broadly relevant, but may lack focus or 2nd organisation, or show limited understanding of the topic, or omit some relevant material. Third Work that shows knowledge of material appropriate to the question, but with deficiencies in understanding, coverage, focus and/or organisation. Fail Work that has serious deficiencies in understanding, coverage, focus and/or organisation, or which does not answer the question asked. The University of Edinburgh Marking Scheme defines four levels of Fail: Marginal Fail (Grade E, 30-39%), Clear Fail (F, 20-29%), Bad Fail (G, 10- 19%) and Bad Fail (H, 0-9%). Marks are to be awarded accordingly. Two or three clear and relevant sentences on the topic may be graded F.

FEEDBACK FOR YOU There will be many opportunities for you to get feedback on your understanding of material in the course. You will get verbal feedback on your answers to tutorial questions from your tutors, and the course problem will be discussed in Tutorial 3. You will get written feedback from your peers on your answer to the course problem and your PeerWise questions and answers. The lecture quizzes will give you feedback when you submit your answers each week. You will see your mark for the data handling test when you submit your answers and general feedback will be provided on Learn after the test.

If you are concerned about your performance in any aspect of the course then you should arrange to meet David Finnegan, the course organiser, or your personal tutor to discuss it.

FEEDBACK FROM YOU We welcome feedback on the course. Feel free to contact David Finnegan at any time, or speak to your Floor-leader in practicals or your Tutor in tutorials meeting and there will be a mid- semester feedback session in Lecture 15. You can find the names of the Programme Representatives for Genes and Gene Action in the “Course Content” section of the GGA site on Learn and you may contact them about any issues that you do not want to raise with the course team directly. There will be a mid-semester feedback session during the lecture on Friday 15th February and the Programme Representatives will attend a Student–Staff liaison meeting near the end of March.

You will be asked to complete a questionnaire at the end of the course where you can tell us what you think of the course and how it may be improved. Bear in mind that while we shall try to act on constructive criticism, it is very difficult to do anything in response to comments such as “I was bored!”.

FREQUENTLY ASKED QUESTIONS Practicals/ Tutorials Q: What should I do if I need to change my practical or tutorial group? A: You should *Complete the Group Change Request form at https://www.edweb.ed.ac.uk/timetabling-examinations/timetabling/personalised-timetables/group- change-request

*Please note that due to the high number of students enrolled on the course this year it may not be possible to move you to your chosen group and you may be asked to arrange to swap groups with a fellow student.

Q: Where can I get model answers to the tutorial questions? A: No standard answers will be given since the purpose of tutorials is for you to understand how to reach the solution. This cannot be achieved by looking at the answer but only by working through the problem and making a record of the answer for yourself. If you wish to check your answers you should contact your peers, your tutor, or another member of teaching staff.

Missed work Q: What should I do if I miss my practical or tutorial group? A: You need to catch up on the missed work so that your own learning is complete. You should do this by speaking to others who have attended and make sure that you understand any problems and solutions that have been discussed. Should any points of difficulty remain you should seek clarification from your tutor or practical leader.

Q: What should I do if I have missed part of the course? A: You need to review all missed work (using the course book, Learn, and your peers) and ensure that you fully understand all the material covered. If any issues remain confusing to you, you should seek help from a member of the course team. If you have missed work due to circumstances beyond your control and you feel that your marks will be adversely affected you should speak to your personal tutor about submitting a special circumstances form.

Assessments: In course Q: How can I get an extension on my in course problem deadline? A: You may be granted an extension if you are unable to submit your answer online before the deadline because of illness or some other special circumstance and you apply to the Student Support Team using the form available at https://www.wiki.ed.ac.uk/display/SBSUndergraduateIntranet/Coursework+Extension+Form

This must be done before the deadline. If you are unable to do this and miss the deadline because of serious circumstances beyond your control then you should speak to your personal tutor about submitting a special circumstances form.

Q: How many questions are in the data handling test? A: There are 6 different sections, each with 2-4 questions pertaining to a set of data.

Q: What happens if I have arranged to be away and cannot make the data handling test? A: Unless there are serious special circumstances preventing your attendance you will fail the course and will need to re-sit in August (and pay the re-sit fee). If you have submitted a special circumstances form then you may be allowed to take the test in August as a “first sit”.

Assessments: Exam Q: How many multiple choice questions will there be in the exam? A: 20

Q: How much should I write in my answer to the exam long answer question? A: There is no specified length but between 1 and 2 pages of the exam book is normal but a longer answer will not get more marks than a shorter answer that addresses the question equally well. No marks will be given for information that is correct but is not relevant to the question.

Q: How should I present my answer to the exam long answer question? A: Unless otherwise directed you should treat it as an essay and, therefore, start by introducing the topic, then deal with the details of the matter in hand before concluding. More importantly it must address the question. A good answer on a related topic will earn little or no marks! An answer that includes a figure that complements your text is likely to gain more marks than the same answer without a figure.

Help about the course Q: Where can I get help? A: For any administrative, organisational or general course-related questions you should email [email protected]. If you are unsure about the content of the course then you should post a question on the Discussion Board, contact the relevant lecturer, your tutor, or the course organiser [email protected]. If you have a question about the practicals then you should ask your demonstrator or contact [email protected].

TUTORIALS

TUTORIAL 1: Recombination, Linkage and Genetic Complementation

1. The following three recessive phenotypes are known in mice:

h hotfoot o obese wa waved.

A mouse of unknown origin, but segregating wild type and mutant phenotypes for each of these three loci, was testcrossed with the following results:

PHENOTYPE NO. OF PROGENY

hotfoot, obese, waved 77 hotfoot, obese 357 waved 343 obese 9 wild type 73 hotfoot waved 11 obese waved 66 hotfoot 64

a) What is the genotype of the mouse of unknown origin? b) What is a test cross? c) Are these three genes linked? d) What was the combination of alleles inherited by the mouse of unknown origin? e) What is the relative order and map distance between these genes? f) Is there any crossover interference? If yes, how much?

2. A crossover has occurred in the bivalent shown below: A B C

A B C

a b c

a b c What is the recombination frequency between A and B observed from this single event? What is the recombination frequency between B and C?

If a second additional crossover was to occur somewhere between A and C, explain which chromatids would be involved and where it would have occurred to produce the following chromosomes;

a) AbC, ABC, aBc and abc b) Abc, aBC, Abc and aBC c) ABC, aBc, Abc and abC d) ABC, ABC, abc and abc

3. You discover a Drosophila with light brown eyes and on a separate occasion you find another with fly with pale cream eyes. You are intrigued to investigate the genetic basis of these phenotypes and make strains homozygous for each mutation. What experiment could you do to establish whether this phenotype is recessive or dominant to wild type?

You find that both light brown and pale cream are recessive to wild type. You now want to know whether these mutations are the same gene or in different genes. What experiment could you do to determine this?

When you cross the two mutant strains you find that all the F1 flies have light brown eyes. A backcross of the F1 to flies with pale cream eyes gives 123 flies with pale cream eyes and 135 flies with light brown eyes. What can you conclude from these results?

TUTORIAL 2: Analysis of Replication Mutants and Restriction Enzymes

1. The restriction enzyme Sau3A recognizes the sequence 5'GATC3' and cleaves on the 5' side (to the left) of the G. (Since the top and bottom strands of most restriction sites read the same in 5' to 3' direction, only one strand of the site needs to be shown.) The single stranded ends produced by Sau3A cleavage are identical to those produced by BamHI cleavage (see Figure), allowing the ends produced by the two types of restriction enzyme to be joined together by incubation with DNA ligase. (You may find it helpful to draw out the product of this ligation to convince yourself that this is true.)

(a) What fraction of BamHI sites can be cut with Sau3A? What fraction of Sau3A sites can be cut with BamHI?

(b) If two BamHI ends are ligated together, the resulting site can be cleaved again by BamHI. The same is true for two Sau3A ends. However, suppose you ligate a Sau3A end to a BamHI end. Can the hybrid site be cut with Sau3A? With BamHI?

(c) What will be the average size of DNA fragments produced by digesting DNA with Sau3A if the sequence of the DNA is random and the ratio of A/T to G/C base pairs is 1:1?

(d) What will be the average size of DNA fragments produced by digesting this DNA with BamHI?

(e) What will be the average size of DNA fragments produced by digesting DNA with Cac81, that recognises the sequence GCNNGC where “N” may be any base, if the sequence of the DNA is random and the ratio of A/T to G/C base pairs is 1:1?

(f) What will be the average size of DNA fragments produced by digestion of DNA with BamHI if the sequence of the DNA is random and that the ratio of A/T to G/C base pairs is 6:4?

2 In a search for Escherichia coli mutants temperature-sensitive for DNA synthesis (dna ts), which were expected to help discover the mechanism of DNA replication, cells were exposed to mutagens and then screened for the ability to grow at 30°C but not at 42°C on broth plates (containing many small-molecule nutrients which E. coli either needs or would otherwise have to make for itself).

a) The use of broth plates avoided the isolation of a whole class of unwanted E. coli ts mutants. Can you see why?

Purified ts mutants were next screened for dnats by growing at 30°C in liquid culture, then shifting the culture to 42°C and continuing incubation. After 60 minutes samples were labelled with 14C- thymidine and 3H-leucine for 5 minutes, and the amounts of 14C and 3H incorporated into large molecule (acid-insoluble) form were determined.

b) How would you expect a dnats mutant to behave? What is the point of labelling the cells with leucine as well as thymidine?

Many dnats mutants were found. All were then mapped into specific genes, by standard mapping and complementation test methods. At least six genes were found in the first searches. Mutants with ts mutations in genes II, III, IV, V and VI showed the behaviour indicated in Figure 1, when 14C-thymidine incorporation into DNA was further studied. Several different ts mutations in gene I, however, led to the results shown in Figure 2.

c) Why are the results in Fig. 1 much as expected? Can you imagine how the function of gene I might be special, in a way that would explain Fig. 2? (This is tricky, so here is a clue: the cells in these experiments are not all at the same stage of the replication cycle when the culture is shifted to 42°C).

Among dnats mutations affecting one of the subunits of DNA polymerase III, a few show the property of producing other mutations (which may be in any gene of E. coli) at an unusually high frequency, when grown at 30°C. Evidently these ts mutations, although not lethal at 30°C, do nevertheless cause some partial abnormality of their gene product at 30°C.

d) What is likely to be the function of the subunit which is affected by these mutations?

TUTORIAL 3: Plasmids and the Course Problem

1. The streptomycin-sensitive (StrS) strain of E. coli, FD1, contains a plasmid carrying an ampicillin resistance gene (AmpR). FDl was mated separately with two streptomycin-resistance (StrR) strains, FD2 and FD3. FD3 contains a plasmid carrying a tetracycline-resistance gene (TetR) while FD2 is plasmid free. Each mating mix was plated on nutrient agar containing ampicillin and streptomycin or ampicillin, tetracycline and streptomycin. The number of AmpRStrR and AmpRTetRStrR cells per ml was calculated. The results were as follows:-

Donor Recipient AmpRStrR AmpRTetRStrR FD1 FD2 107 0 FD1 FD3 5 x 106 25

a. What can you conclude from the results? b. What might be the properties of any plasmid present in the AmpRTetRStrR cells?

2. Model answer, feedback and discussion of the course problem.

Please bring to the tutorial a copy of your answers to the course problem.

Your tutor will work through the problem with you and this will be your opportunity to ask questions about the model answer to the problem. You should also clarify anything that remains unclear to you about what you have learned from the problem.

You will have two weeks to mark the answers to the course problem that were submitted by two of your peers, as well as your own. Papers will be assigned to you randomly and you will mark them using the PeerMarking function of learn where marking guidelines will also be available. It is important, therefore, that you fully understand the answer to the question. The PeerMarking exercise is intended to provide you with an understanding of how to approach problem questions in the exam as well as in future courses, and completion of the PeerMarking exercise (both your peers and your own script) contributes to your mark for the the course problem. The answers that you should mark, together with the marking guidelines, will be available via PeerMark on Friday March 6th and you must complete your marking by 4.00pm on Friday March 20th.

TUTORIAL 4: The Genetic Code and Pedigree analysis

1. The general nature of the genetic code was determined largely by a brilliant set of genetic experiments carried out by Crick, Brenner and colleagues in 1961, analysing mutations in the rII genes of coliphage T4. Inter alia, they concluded that frameshift mutations could be caused by insertion or deletion of (probably single) base pairs, and (in favourable cases) "corrected" by (respectively) deletion or insertion of single base pairs at a nearby point in the same gene, such as to restore the frame of correct translation (and thus synthesis of a functional protein).

These authors were unable to verify their interpretations directly, because no one had succeeded in purifying the rII proteins; the sequence of amino acids in the wild-type could not be determined and compared with, for example, a double mutant. Later George Streisinger and colleagues, studying similar "suppressed frameshift" mutations in the lysozyme gene of T4, were able to show that the effects on protein sequence were exactly of the nature predicted by the rII workers. In a specific case, a sequence within the lysozyme protein was changed as follows. (The amino-terminal {N} side of each fragment is on the left.) Note that it was already known that protein chains are synthesized from the amino towards the carboxyl end.

Wild-type (normal): thr-lys-ser-pro-ser-leu-asn-ala-ala-lys

Double mutant: thr-lys-val-his-his-leu-met-ala-ala-lys

Using these results, and the genetic code table (inside front cover) work out the sequences of the 30 nucleotides of wild-type and mutant mRNA encoding the two polypeptides. Your work will identify the two mutations which generated the functional double mutant. Streisinger et al already knew from genetic evidence that only two mutations were involve

SECOND POSITION FIRST THIRD T/U C A G phe ser tyr cys T/U phe ser tyr cys C T/U leu ser stop stop A leu ser stop trp G leu pro his arg T/U leu pro his arg C C leu pro gln arg A leu pro gln arg G ile thr asn ser T/U ile thr asn ser C A ile thr lys arg A met thr lys arg G val ala asp gly T/U val ala asp gly C G val ala glu gly A val ala glu gly G

2. DNA studies have been performed on a family with an inherited disease. The pedigree given shows only whether each individual is affected or unaffected with the disease. A DNA sample from each member is digested with the restriction enzyme Taq1 and run on an electrophoretic gel. A Southern (DNA blot) analysis is then performed using a radioactive probe consisting of a portion of human DNA cloned in a bacterial plasmid.

Pedigree

I.1 I.2 II.1 II.2 II.3 II.4 II.5 II.6 II.7 II.8 II.9

5kb

3kb 2kb

a) Consider just the pedigree data. What can you conclude about the possible mode of inheritance? b) Consider just the DNA blot data. What can you say about the human DNA used for the probe? Draw a simple diagram to show the distribution of TaqI sites and the position of the probe within this region of human DNA. c) Consider both the pedigree and the Southern DNA blot data together. What is the relationship between the DNA variation and the gene for the disease? Does this help you to decide about the mode of inheritance? d) How do you explain the last son (individual II.9)? Draw a diagram of the relevant chromosomal regions for each parent on level I and for individual II.9.

PRACTICAL SYNOPSIS

1. Genomic DNA preparation for Polymerase Chain Reaction (PCR); Bacterial conjugation (interrupted mating of E. coli) and Three point crosses. (1) You will prepare a genomic DNA sample from root hair (for use in a PCR in Practical 2). (2) You will perform an interrupted mating (conjugation) experiment using an Hfr (High frequency recombination) strain and an F minus strain of Escherichia coli to allow you to construct a simple genetic map of some auxotrophic genetic markers in Practical 2. (3) A simulated three point cross in Neurospora will provide data for mapping three loci.

2. PCR (part 2); Yeast genetics & complementation; Preparation of plasmid DNA and Bacterial conjugation (part 2). (1) You will set up a PCR using the genomic DNA you prepared in Practical 1. (2) You will test yeast (Saccharomyces cerevisiae) strains for their auxotrophic requirements and mating types. Your results will allow you to set up yeast matings to perform a complementation test to determine whether particular auxotrophic mutations are in the same or different genes. (3) You will prepare plasmid DNA from an E. coli strain which you will analyse in subsequent practicals. (4) Following on from Practical 1 you will analyse the data from your interrupted mating experiment and construct a simple genetic map of the auxotrophic genetic markers used.

3. Gel electrophoresis of PCRs (part 3); Restriction enzyme digestion of plasmid DNA (part 2); Yeast genetics & complementation (part 2); Three exercises: Genetic problem, Lac operon and Size estimation of DNA fragments. (1) You will carry out agarose gel electrophoresis on your PCR samples set up in Practical 2. (2) You will digest the plasmid DNA prepared in Practical 2 with restriction enzymes which you will analyse in later practicals when you will construct a plasmid map. (3) You will analyse the data from the yeast complementation test you set up in Practical 2. (4) There is a genetics problem for you to solve in the practical. (5) An exercise on Learn asks you to analyse the -galactosidase activity in a number of E.coli strains carrying mutations in the lac operon. You should do this in your own time, but ideally before Practical 4. (6) You will estimate, in your own time, the size of the DNA fragments on a sample gel (provided in the coursebook) using a standard curve. Bring your estimates to Practical 4 where you will use them to construct a plasmid map.

4. Gel electrophoresis of restriction digests of plasmid DNA (part 3); Analysis of yeast complementation test (part 3); Bacteriophage T4 rII locus intragenic recombination; Analysis of the D1S80 region of human DNA (PCR part 4) and Construction of a plasmid map (part 2). (1) You will perform gel electrophoresis on the restriction digests of the plasmid DNAs from Practical 3 (you will analyse the data generated and construct plasmid maps in Practical 5). (2) You will analyse the results from the yeast complementation test set up in Practical 3. (3) You will be given two T4 rII mutants to test for lytic growth in two strains of Escherichia coli (B and K12). You will perform a mixed infection of B strain with the two T4 mutants and screen the products for recombinants by plating on K12 strain.

(4) Supporting the lac operon exercise on Learn, plates of E. coli strains with different mutations in the lac operon will be available to study. (5) Using the gel results from Practical 3, you will estimate the size of your PCR derived DNA fragments using a standard curve and estimate the number of repeats present; class data will be collated. (6) Using your DNA fragment size estimates (from the gel in the coursebook) you will construct a restriction map, in anticipation of the procedure required in Practical 5.

5. Analysis of bacteriophage T4 rII locus intragenic recombination (part 2); Analysis of plasmids A & B (part 4) and Gene mapping in humans. (1) You will analyse and interpret the results of the bacteriophage T4 rII locus intragenic recombination experiment set up in Practical 4. (2) Photographs of your plasmid DNA restriction enzyme digestion gels will be available and you will construct restriction maps of plasmids A & B using these data.

(3) You will analyse genetic data from human family pedigrees with the aim of interpreting the data and mapping human genes

P1 P2 P3 P4 P5 Exercise Jan 15 /22 / 23 Jan 29/ Feb 5 / 6 Feb 12/ 26/ 27 Mar 4 /11 / 12 Mar 18/ 25 / 26 PCR Part 1 (set up Part 2 (set up PCRs) Part 3 (load gels) Part 4 (analyse results – std DNA extraction) curve and size estimation) Interrupted mating Part 1 (plate out) Part 2 (count of E. coli (bacterial colonies & plot conjugation) graph) 3 point mapping Analyse data provided Yeast genetics & Part 1 (plate out) Part 2:( a) analyse growth Part 3 (Analyse and interpret complementation requirements & mating complementation test) types b) set up complementation test) Plasmid prep/digest Part 1 (prepare Part 2 (set up restriction Part 3 (load gels) Part 4 (analyse results- std and mapping plasmid) digests on plasmid) curve, estimate sizes and construct plasmid maps – answer questions Neurospora Analyse data provided Genetics problem Sample data Homework: Plot std curve Construct plasmid map plasmid mapping & estimate sizes in your own time ready for P4 Lac operon Homework: Learn exercise Observe plates and confirm your interpretation Intragenic Part 1 (plate out) Part 2 (analyse and recombination interpret results) bacteriophage T4 Genetic data (sex Analyse data and answer linked) exercise questions Human pedigree Analyse data and data exercise construct map of chromosome region

PRACTICAL 1

GENOMIC PCR EXPERIMENT-

ANALYSIS OF REPETITIVE DNA AT THE HUMAN LOCUS D1S80

Introduction

Eukaryotic genomes contain a large number of repetitive sequences; D1S80 is a locus on human chromosome 1 that consists of a series of a repeating 16 bp sequence. It belongs to the so called Variable Number of Tandem Repeats (VNTRs) and is a minisatellite sequence. Because the number of repeated sequences is variable between alleles, the length of this locus is variable (Fig. 1). At the D1S80 locus, most individuals have alleles containing between 14 and 41 repeats, which are inherited in a Mendelian fashion from the maternal and paternal copies of chromosome 1. This, and similar loci in the human genome, are used in genetics (population studies, linkage to various diseases - diagnostics) and, of course, forensic science. One of the possible ways to analyse such short repetitive regions is to amplify them by PCR - this method is also called Amplified Fragment Length Polymorphism - AFLP. In this practical, each student will prepare a sample of their own genomic DNA which will be used in a subsequent PCR reaction to allow comparison of the length of D1S80 products to those of other members of the class.

Figure 1. Schematic map of the D1S80 locus. Positions of primers are indicated. Note that the first repeat has only 14 base pairs (bp). The 5’ and 3’ primers consist of 28 and 29 nucleotides respectively. The number of bp between the primers and first or last repeat region are indicated.

The polymorphic region, which is amplified with the primer set, is not associated with a human disease allele and would not be sufficient to identify individuals by genotyping. We hope that you will supply your own hair follicle for this experiment, but a random sample of human DNA will be supplied if necessary.

Isolation of Genomic DNA from Hair Roots

In this very short session you will isolate your genomic DNA from a hair root. The DNA obtained will be used in the next session to amplify the D1S80 locus by PCR.

Materials supplied:

DNA isolation buffer: (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 35 mM DTT, 1.5 mM MgCl2, 0.45 % Nonidet P40, 0.45 % Tween 20, 0.25 mg/ml Proteinase K.) 1.5 ml snap cap tubes. 32

Procedure:

1. Pluck a hair (or two) so that it includes a hair root. Note: a hair root must be visible otherwise you will not have any DNA in your sample. 2. With scissors, cut about a 5 mm long portion of the hair with the root and put it into a pre-labelled 1.5 ml locking-cap tube. The label gives your group number and individual ID, please take a note of these so that you will be able to identify your sample in the next practical (you can add your initials to the side of the tube if you wish). 3. Add 200 l of DNA extraction buffer and spin the tubes for few seconds in a microfuge to bring the hair root and liquid down to the bottom of the tube. Ensure that the hair root is at the bottom of the tube and is covered with buffer. 4. Place your sample tube in the appropriately labelled box on the side bench. 5. Incubate the sample at 56oC for 3 hours with shaking (done for you by technicians). 6. Incubate the sample at 95oC for 10 minutes. Store at 4oC till the next practical session (done for you by technicians).

INTERRUPTED MATING EXPERIMENT WITH ESCHERICHIA COLI

The purpose of this practical is to investigate transfer of auxotrophic markers between two strains of E. coli by making use of the bacterial conjugation system. The two strains will be an Hfr donor strain and a multi-auxotrophic F- recipient.

DONOR Strain P4X: Hfr Str S arg + pro + leu + RECIPIENT Strain AB1157: F – Str R arg – pro – leu -

Make sure you understand what the strain genotypes mean.

You will work in groups of four for this experiment.

Material provided: All agar plates provided contain minimal agar, streptomycin and supplements but lack an amino acid as indicated. The plates are colour coded with a red, blue or green stripe; you should label these A, B and C respectively.  5 plates (A – red stripe) NO PROLINE  5 plates (B – blue stripe) NO LEUCINE  5 plates (C – green stripe) NO ARGININE  1 sterile 15 ml screw capped conical tube  1 bottle of sterile 50 mM sodium phosphate buffer pH7 / 0.5 % NaCl on ice (referred to as sodium phosphate buffer later)  5 sterile bijou bottles  10 sterile microfuge tubes  2 sterile 5 ml tips  Sterile 1 ml tips  Sterile 0.2 ml tips  5 sterile cell spreaders  5 ml Hfr donor cells in bijoux bottles  5 ml F- recipient cells in bijoux bottles Please note that some of these items may be on the front and / or side benches.

POINTS TO REMEMBER WHEN PERFORMING THE EXPERIMENT:  Keep the lids on the pipette tip racks when not being used. 33

 Keep the lid on the box of microfuge tubes closed when not in use.  Leave the lids on plates at all times unless you are using that plate.  Work fast while the lid is off the plate and the tops are off the tubes.

Why do you think it is important to follow these instructions?

METHOD Note that there are five time points in this experiment: 0, 20, 40, 60 and 80 minutes:

Before you begin the experiment, do the following:  Pipette 0.9 ml of sodium phosphate buffer into each of the 5 bijou bottles and label them 0, 20, 40, 60 or 80 (minutes) and place on ice  Label the microfuge tubes and pipette 0.9 ml of sodium phosphate buffer into each of the 10 microfuge tubes (2 for each time point) and place on ice  Label the base of your plates with: A, B or C (following the instructions above), your ID (i.e. your group number on the rack provided on your bench) and time point (0, 20, 40, 60 or 80 min)

You can now begin the interrupted mating experiment:

1. At time zero, add 4 ml of Hfr donor and 4 ml F- recipient cells to the sterile 15 ml conical tube, invert the tube a few times to mix.

2. Immediately pipette 0.1 ml from the mating mixture and dilute it into the 0.9 ml sodium phosphate buffer in the bijou bottle (this is time 0).

3. Replace the remaining mating mixture to a non-shaking 37oC water bath to allow mating to continue.

4. Vortex the sample in the bijou bottle intermittently (i.e. regularly placing the bottle on and off the vortex) for 1 minute.

5. Pipette 0.1 ml from the bijou bottle and add to microfuge tube 1 containing 0.9 ml sodium phosphate buffer. Mix the contents by vortexing briefly.

6. Pipette 0.1 ml from microfuge tube 1 and add to microfuge tube 2 containing 0.9 ml sodium phosphate buffer. Mix the contents by vortexing briefly

7. Pipette 50 μl of cells from microfuge tube 2 onto each correctly labelled plate A and B and pipette 50 l of cells from microfuge tube 1 onto the correctly labelled plate C.

8. Spread the cells evenly around the plate using a cell spreader (the same cell spreader may be used per time point so long as you spread plates A and B first before spreading plate C – why do you think that this is important?). Keep spreading until all the liquid has been absorbed and then place the plates into the rack provided. Please note it is not necessary to use a Bunsen burner.

9. Discard the bijou bottle, microfuge tubes and cell spreader for each time point into the waste bag, this will avoid confusion with the samples from the other time points.

10. For each of the other time points repeat step 2 to 8 remembering to invert the tube once before taking the 0.1 ml aliquot. 34

11. The plates will be incubated at 37C for 48 hours and then stored at 4C until the next practical.

QUESTIONS

1. What is a bacterial plasmid? 2. What is an Hfr bacterial strain? 3. What is an F- bacterial strain? 4. In the above experiment why does the Hfr need to be streptomycin sensitive? 5. What is an auxotroph? 6. In the above experiment what is the purpose of the vortexing in step 4? 7. a) How many dilutions were performed in steps 1 to 6? b) what were the individual dilution factors c) what was the final dilution factor in microfuge tubes 1 and 2?

NEXT PRACTICAL

You will count the colonies on plates A, B and C, calculate the number of exconjugants/ml and plot this against time prior to interruption of mating.

THREE POINT MAPPING

You will work in groups of four for this exercise.

On each bench you will find a plastic container with laminated cards on which appear three genotype symbols:-

arg- = arginine requirement (arg+ = wild type)

trp- = tryptophan requirement (trp+ = wild type)

col- = colonial growth (col+ = wild type)

Imagine that these are the meiotic products obtained when a triple heterozygote undergoes meiosis in Neurospora. Each paper represents an ascospore.

Sort them according to genotype and count the number in each group. How many groups do you expect? How many did you find? Use your data to infer the likely genotype of each haploid parent. Use your data to calculate the percent recombination between the three genes. Draw a genetic map and calculate the coefficient of coincidence (CoC) and interference value (I) for your data.

observed double recombinants CoC = ------I = 1 - CoC expected double recombinants

Please return the papers to the container so they may be used by the next class.

Please fill out the class data sheet for this exercise. The pooled class data for this exercise will be made available on Learn. You should access these data and calculate the coefficient of coincidence and interference value for them.

PRACTICAL 2

GENOMIC PCR EXPERIMENT –PART 2- ANALYSIS OF REPETITIVE DNA AT THE HUMAN LOCUS D1S80

Setting up the PCR

Today you will use the PCR technique to amplify alleles of the D1S80 locus using the genomic DNA samples prepared in the last session.

Materials supplied:

 Each group of 4 will be provided with Eppendorf tubes (microfuge tubes) containing: Mastermix containing PCR buffer (GoTaq Green), dNTPs, DNA primers, Taq polymerase and ddH2O at concentrations appropriate to achieve a final concentration of 1 M for each DNA primer, 400 M for each dNTP and 1.5 units of Taq polymerase in the PCR set up as described below).  Your genomic DNA (template DNA) isolated in Practical 1  Thin-walled 200 l PCR tubes (pre-labelled) will also be provided

Procedure:

1. Vortex your DNA sample briefly and then spin for 1 minute at full speed in a microfuge to pellet all residual debris. 2. You will use the pre-labelled 200 l PCR tube (labelled with your group ID and ‘A, B C or D’). You should take a note of which tube you used for your sample. 3. Set up the PCR reaction ON ICE as follows: Take care to ensure that your pipetting is accurate.

Mastermix 40 l DNA sample 10 l

4. Vortex the tubes briefly and spin for few seconds in a microfuge with a transparent coloured domed lid. Note: please ensure you read the operating instructions for this microfuge before use. 5. Place your PCR tube in the appropriately labelled rack on ice provided on the side bench. 6. The tubes will be put in the PCR machine and the PCR will be run using the following conditions:

1 cycle 95oC 4 min

35 cycles 95oC 30 sec 64oC 30 sec 72oC 90 sec

1 cycle 72oC 5 min

7. After the run your samples will be stored at -20oC until the next session.

8. Return the tube containing the remainder of your DNA sample (template DNA) to the storage rack from which you first collected your tube.

CLASSIFICATION OF AUXOTROPHIC MUTANTS IN YEAST (Saccharomyces cerevisiae)

You will work in groups of four for this experiment.

This experiment will occupy three weeks. You will use a simple method to make yeast diploid and will form diploids between different yeast strains. You will then interpret the results and draw conclusions on the genes involved in the auxotrophic strains you’ve tested.

Each group will be given a series of haploid yeast auxotrophic mutant strains. Each strain has a specific growth requirement, which means that minimal medium (sugar + essential salts + agar) plates need to be supplemented with a substance or substances (single amino acids or nucleic acid precursors) if the strain is to grow. In this experiment each requirement can be assumed to be due to mutation in a single gene locus.

Determine the growth requirements for each mutant strain.

You are provided with 5 strains of yeast (A, B, C, D and E). These need to be tested to determine their growth requirement[s] and to do this you are provided with 7 different types of growth media. These are listed in the table below where  shows that the substance has been added to the medium.

Plate Arginine Histidine Leucine Tryptophan Adenine 1      2 ZERO     3  ZERO    4   ZERO   5    ZERO  6     ZERO 7 ZERO ZERO ZERO ZERO ZERO

Medium type 1 has 5 different supplements; types 2 to 6 each has one of these supplements omitted. Medium 7 has no supplements.

First label each of plates 1-7 on the bottom [NOT the lid! Why?], with the letter of strain to be tested and your group number that is on the rack on your bench (e.g. 14) as shown diagrammatically below:

A E B 14

D C

Now take strain A, re-suspend the pellet of yeast cells by thoroughly shaking the container, and then using one of the sterile plastic loops provided inoculate EACH plate with strain A in the appropriate place on the plate. Repeat this procedure for each strain TAKING A NEW LOOP FOR EACH STRAIN (i.e. you will use a total of 5 loops when setting up this part of the experiment). Make sure the liquid suspensions are confined to the appropriate place on the agar and do not run into one another. It might be necessary to allow the liquid to absorb into the agar between inoculations.

Determine the mating type of your mutants

Haploid yeast strains exist as either a or  mating types. You can only form diploids – and hence carry out a complementation test - between strains of opposite mating type. To decide which combinations of your strains will form diploids you therefore need to determine their mating type. Strains D and E are of opposite mating type and so you can use these to test the others and determine the mating type of all the strains.

You are provided with a second plate of minimal media [type 7]. Use this plate to determine the mating type by proceeding as follows - LOOK AT ALL THE DIAGRAMS BELOW BEFORE STARTING:

Label the bottom of the plate with bench number and strains to be tested as shown below.

A B C D

Now re-suspend the strains as before and using a loop, streak out strain D and [USING A FRESH LOOP] strain E as shown diagrammatically below. 38

A B C D

Now streak out strains A, B and C by cross streaking each against strain D (use a fresh loop for strains A, B and C). This is shown diagrammatically below:

A B C D

Now repeat the operation for strain E when your plate should look like this:

Note that you will use a total of 8 loops to set up this plate.

A B C D

The final diagram shown next summarises the whole process:

Make sure you have mixed the two strains in the region marked with diagonal stripes; it is in this region, where the two strains are mixed, that diploids will form if the strains are of opposite mating type.

The plates used in the experiment will be incubated at 30oC. Place the plates in the wooden rack provided.

QUESTIONS: Think about what you have done in this experiment by considering the following:

1. Will you be able to deduce the phenotypes of the strains from the growth response you observe on the different media?

2. Will you be able to deduce the genotypes of the strains from the growth response you observe on the different media?

3. If you are told the mating type of one of the strain, will you be able to deduce the mating type of the others?

MAPPING RESTRICTION SITES IN PLASMID DNA Introduction The aim of this practical is to enable you to obtain technical experience in (1) isolation of plasmid DNA from E.coli cells and (2) construct a simple restriction enzyme map of plasmid DNA.

Recombinant DNA technology has enabled sequences to be cloned into plasmid vectors and amplified in E.coli host cells. The plasmid DNA used in such cloning procedures is a circular molecule, such as the general cloning vector pUC18 shown in Figure 1 and contains a selectable marker, in this case the -lactamase gene which confers resistance to ampicillin (ampR). As with all cloning vectors, it also has a polylinker region which contains unique recognition sequences for a variety of restriction enzymes called the Multiple Cloning Site (MCS) and these sites are used to clone fragments of DNA which have compatible ends. Some of these restriction enzyme sites in pUC18 are shown at the bottom of Figure 1.

LacZ Figure 1 MCS

pUC18 2686 bp

ampR Replication origin

Polylinker MCS

HindIII PstI XbaI BamHI SacI EcoRI -AAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTC- -TTCGAACGTACGGACGTCCAGCTGAGATCTCCTAGGGGCCCATGGCTCGAGCTTAAG-

Human -globin gene clones A PstI fragment from human genomic DNA containing the -globin gene was inserted into the PstI site in the polylinker region of the pUC18 plasmid vector. This was achieved by joining together the compatible PstI ‘sticky’ ends of the vector and insert using DNA ligase to form the recombinant circular plasmid containing the -globin gene insert (Figure 2). The pUC18 vector DNA was pre-treated with phosphatase after PstI digestion to prevent self-ligation of the vector (as these would not contain an insert). The ligated molecules were then transformed in to E.coli DH5 cells and recombinant clones were selected by plating onto agar plates containing ampicillin. Only cells which contain the circularized

plasmid vector which contains the ampR gene will grow; all other cells will be killed by the antibiotic.

Figure 2

PstI PstI PstI DNA PstI Ligase

PstI Recombinant pUC18 + plasmid PstI - globin gene

Two different recombinant clones were derived from this experiment (A and B) both of which contained the -globin insert. The object of this practical is to isolate each of these recombinant plasmids from cultures of E.coli, subject the plasmids to restriction enzyme analysis and produce a simple restriction map of each clone. By comparing the restriction maps, you should be able to determine the structure of each clone and why the maps are different.

Isolation of Plasmid DNA from E.coli cells

You will work in groups of four; one pair of students will isolate DNA from clone A and the other from clone B and you will share your results.

Safety Note: Gloves must be worn at all times during the preparation of the plasmid DNA as the reagents used are potentially harmful.

Material supplied:

You are provided with 6 coloured tubes of buffers labelled: P1 (on ice) P2 – contains sodium hydroxide N3 – contains guanidine hydrochloride and acetic acid PB - contains guanidine hydrochloride and isopropanol PE EB Tubes of E. coli culture A or B

Each pair are provided with a spin column and collection tube as well as Eppendorf (microfuge) tubes

Procedure: 1. You are provided with two labelled microfuge tubes (approximately 1.5 ml per tube) of E.coli culture A or B.

2. Place the microfuge tubes in the microcentrifuge and spin for 3 mins to pellet the cells. Note: Make sure that there is another tube in the slot opposite, so that the rotor is balanced – make sure you take this precaution for every centrifugation step performed.

3. Remove the supernatant with a pipette and place into the liquid waste container. Mix the P1 buffer before adding 250 l of it to the cell pellet. Resuspend the pellet by vortexing or pipetting up and down using the appropriate pipette and tip. Note: Make sure that there are no clumps of cells remaining.

4. Add 250 l of Lysis buffer P2 and mix by gently inverting the tube 4-6 times - do not vortex. The solution should become clear, blue and viscous. The blue colour should become uniform when the solutions are mixed properly. Note: do not let the lysis reaction proceed for more than 5 mins. [This step causes lysis of the cell walls and denatures chromosomal DNA, plasmid DNA and protein].

5. Add 350 l of the Neutralising buffer N3 and mix immediately by inverting the tube 4-6 times. The blue colour should now disappear when mixing is complete. [This step neutralises the alkaline lysis buffer and causes the denatured protein and chromosomal DNA to precipitate, whilst the smaller plasmid DNA renatures and stays in solution.]

6. Place the labelled tube in the microcentrifuge. Spin at top speed for 10 mins. [This will pellet the unwanted protein / chromosomal DNA and the plasmid DNA will remain in the supernatant]

7. Make sure that the spin column is placed within the collection tube. Remove the supernatant from your centrifuged sample and add this to the spin column. Label the spin column with your I.D. and A or B.

Note: the spin column does not have a cap, this is one of the rare occasions that you spin uncapped tubes in a microcentrifuge.

Spin column (blue)

Silica membrane

Collection tube

8. Centrifuge for 1 min. [The plasmid DNA binds to the silica membrane in the column in the presence of high salt and the reaction solution containing contaminants passes through.]

9. Remove the column, dispose of the liquid from the collection tube and replace the column again. Add 500l wash buffer PB and spin in the microcentrifuge for 1 min.

10. Remove the liquid from the collection tube as before and replace column. Add 750l of wash buffer PE to the column and spin in the microcentrifuge for 1 min.

11. Remove the liquid from the collection tube as before and replace column. Spin in microcentrifuge for 1 min to remove any traces of ethanol in the wash buffer. [Both wash steps are used to clean the plasmid DNA which is attached to the membrane.]

12. Remove the column and place it into a clean, labelled, capless, microfuge tube. Add 50 l of elution buffer (EB) to the centre of the silica membrane (without touching the membrane) then place the assembled column / tube in the microcentrifuge. Centrifuge for 1 min. [The plasmid DNA is eluted from the membrane into the low-salt elution buffer.]

13. Discard the column and transfer the plasmid DNA from the capless tube into the clean pre-labelled microfuge tube provided. The label gives your group ID and indicates plasmid A or B.

14. Place the tubes containing the plasmid in the appropriately labelled rack on the side bench of the lab. This plasmid DNA is ready for restriction enzyme analysis in the following practical.

INTERRUPTED MATING EXPERIMENT --- PART 2 1. Count the colonies which have grown on plates A, B and C from all five time points which you set up in Practical 1. Seek advice from your demonstrator if required. Fill in the table below: Number of colonies per plate Microfuge tube Time (minutes) Plate used (1 or 2) Medium 0 20 40 60 80 A Minus Pro B Minus Leu C Minus Arg

2. From these results, calculate the number of StrR exconjugants per ml of F- cells. You will need to take into consideration the dilutions you performed in the experiment in Practical 1 (remember you considered this when answering question 7 in Practical 1). Ensure that you check which microfuge tube (1 or 2) you took the aliquot from for spreading on each plate (A, B and C) in practical 1 – you can record this in the table above. You may want to check your calculations with your demonstrator. Enter your results into the table below: Number of StrR exconjugants/ml Time (minutes) Plate Medium 0 20 40 60 80 A Minus Pro B Minus Leu C Minus Arg

3. Plot a graph of number of exconjugants per ml against time prior to interruption of mating, i.e. 0, 20, 40, 60 and 80 minutes for each series of plates. Be careful how you join the points on the graph. Think of the bacterial chromosome entering the recipient cell with genes at different points along its length. Try to anticipate the type of curve you should get from a gene near the origin of transfer compared to a gene located further from the origin. Try to decide whether to join the points with a smooth curve which passes through the graph origin or should the slope be projected to cut the abscissa? Can you get an estimate of when Pro+, Arg+ and the Leu+ markers start to enter the recipient cell? How do you measure this from your graph?

4. Draw a simple map with the origin of transfer and the three markers on it. What units of map distance will you use? **Please add your results to the class data sheet**

5. Here are some data FROM ANOTHER EXPERIMENT using DIFFERENT Hfr and F- strains but USING THE SAME PROTOCOL, except ALL aliquots for plating out were taken from microfuge tube 1. Calculate the number of exconjugants per ml and then plot these data as before but on a separate graph and repeat the process of drawing a map for these three markers.**Please bring this sample data graph to your next practical**** Number of colonies ------Time (mins) ------Medium: 0 10 20 35 50 80 minus PROLINE 0 45 130 160 163 165 minus LEUCINE 0 0 22 75 82 84 minus ARGININE 0 0 0 10 23 26

QUESTION: Why do the curves flatten out at different levels? 45

PRACTICAL 3

GENOMIC PCR EXPERIMENT – PART 3

ANALYSIS OF REPETITIVE DNA AT THE HUMAN LOCUS D1S80

Agarose gel electrophoresis of PCR samples

In this session you will separate the PCR products on 1.5% agarose gels. The resolution of the gels should be sufficient to distinguish alleles which differ by a few [16 bp] repeats.

Materials supplied:

1.5% agarose gels in 0.5x TBE buffer (44 mM Tris, 44 mM Boric acid, 0.5 mM EDTA)

Procedure: 1. Briefly spin your PCR tube in a microfuge with a transparent coloured domed lid. Note: Please ensure that you read the operating instructions before you use this. 2. Using a Gilson P20 pipette, load 20 l of your PCR reaction onto a prepared 1.5% agarose gel (the demonstrators will load a DNA size marker). The PCR mix already contains a reagent which causes your sample to sink into the loading well. Return the tube containing the remainder of your PCR reaction to the storage rack. 3. Gels will be run at 100 V in 0.5x TBE buffer until the yellow dye is near the bottom of the gel (a technician will set the gels running). The PCR buffer contains two dyes; the yellow dye migrates slightly faster than the DNA primers used in the PCR reaction (<50 bp) the blue dye migrates at about the same distance as a 3-5 kilobase (kb) pair DNA fragment.

Gels will be stained and photographed by lab technicians. A photograph of your gel will be given to you in the next practical session and will also be available on the course Learn site.

Remember to record where you load your PCR sample (i.e. which gel and which well) and where the DNA size marker was loaded.

MAPPING RESTRICTION SITES IN PLASMID DNA –PART 2

Mapping Restriction Sites in DNA

Type II restriction endonucleases recognise specific sequences in DNA and cleave the DNA within this recognition sequence. The recognition sequences are known as restriction sites. It is possible to construct a physical map (restriction map) of DNA showing the positions of different restriction sites and the (approximate) distances between them. Normally restriction maps are constructed from electrophoretic data on the fragment sizes produced when a DNA of interest is digested with two restriction enzymes together and separately. Of course if the DNA of interest has been sequenced the restriction map may be constructed by scanning the DNA sequence for known restriction sites. Restriction maps are invaluable for dissecting and comparing DNAs for which there is no sequence information and planning cloning strategies.

Treatment of a pure sample of a single DNA molecule (e.g. plasmid, virus or chromosome) with a restriction enzyme will produce a precisely defined set of fragments whose number depends on the number of restriction sites for that enzyme in the DNA (of course the longer the DNA molecule, the more complex the mixture of different DNA molecules produced after digestion with a restriction enzyme and the more difficult it is to produce a map). 46

Restriction Enzyme Digestion of Plasmid DNA In the first part of this study (in Practical 2) you isolated plasmid DNA from two E.coli strains A and B which each contain plasmids with an -globin gene insert. In order to produce a restriction enzyme map of each of these clones, DNA samples are treated with single restriction enzymes, EcoRI or HindIII, or a combination of both EcoRI + HindIII (double digest). After incubation, the reaction products will be analysed by gel electrophoresis. Again work in groups of four; one pair will subject the DNA from clone A to digestion with restriction enzymes, the other pair will use clone B (you will share results).

Procedure:

1. Using a Gilson pipette, set up the following reaction mixtures for one of your plasmid samples A or B. Note: use a fresh sterile tip for each addition and add reagents in the order given.

(i) 12 l double distilled water 4 l plasmid DNA 2 l 10x restriction buffer H 2 l EcoRI

(ii) 12 l double distilled water 4 l plasmid DNA 2 l 10x restriction buffer M 2 l HindIII

(iii) 10 l double distilled water 4 l plasmid DNA 2 l 10x restriction buffer Y 2 l HindIII 2 l EcoRI

(iv) 13 l double distilled water 7 l plasmid DNA

2. Label each tube with the plasmid used (A or B) and type of digest (E, H, E+H or C [for control no-enzyme]).

3. Ensure the reaction mixture is well mixed by closing the lid firmly and briefly vortexing the tube (2 sec).

4. Spin the tubes in the microfuge for 10 sec at full speed in the microfuge.

5. Place the tubes in the bag which is already labelled with your group number and place them in the appropriately labelled tray on the bench at the side of the lab.

6. The reactions will be incubated at 37oC for 90 minutes before being placed at -20oC until the next practical session.

YEAST EXPERIMENT --- PART 2

This week, working in your group of four, you will record the results from the growth and mating type tests and work out the growth requirements and mating type of each strain. You will then carry out further tests to work out whether two strains that have the same growth requirement have mutations in the same or different genes.

The plates that you set up last time are returned to you. Examine your tests for growth requirements of the five strains A-E and record your results in your practical book using a table like the one below.

Strain Growth Requirements Genotype Mating Type

A B C D E

Examine the mating tests you set up last time and determine the mating type of each strain. Record the pairs of strains which formed diploids and deduce the mating type of each strain assuming that strain D is mating type a.

You should see that three strains each require histidine for growth but each has an additional growth requirement. Are these histidine requirements caused by mutations in the same gene? To obtain the answer to this question you need to set up a complementation test.

You are provided with two plates of agar. One is a plate of minimal medium containing no supplements; yeast that can grow on this medium must be prototrophic. The second plate is supplemented with histidine and so can support the growth of histidine auxotrophs.

To set up the complementation test, first check with your demonstrator that you have correctly identified the three strains that require histidine for growth, and then decide which pairwise combinations of these three strains will form diploids. Bear in mind their mating types when deciding this. Label the plates with your group number and with the identification letter of the strains to be tested. Your demonstrator will supply you with the yeast strains that you have chosen to use in the complementation test. Now, using the same cross-streaking method that you used last time, cross-streak the strains you wish to test, remembering to resuspend the yeast by briefly vortexing the tubes before you begin (check that the cell pellet has resuspended). Carry out the cross-streaking on both plates so that any pair of strains you test is tested on both types of medium.

In order to help with interpretation of your results next time, you will find it helpful to write down now the genotypes of the haploid parents that you have crossed streaked and the diploid strain you hope to make. You should use standard three letter abbreviations for the genes such as HIS+ for the non-mutant allele and his- for the mutant allele and ADE+ and ade- for the alleles of adenine gene.

QUESTIONS: Test your understanding of complementation and recombination by trying to answer the following questions:

1. Why do you need both minimal and histidine plates? Why not just minimal plates? 2. Why do you need double auxotrophic strains for this test? Why not just his- strains? 3. You have produced diploid strains in this practical. How are haploid strains formed from diploids? 4. If you were able to produce haploid strains from your diploids what types of auxotrophs would you expect to see?

5. You have crossed streaked two yeast strains to form a diploid and the conclusion is drawn that, if growth occurs, the strains must have complemented. Explain, in general terms, why the diploid should grow if the mutant strains complement one another. Why is growth in this case the result of complementation and not recombination? If strains capable of growing on minimal medium were produced as a result of recombination rather than complementation, what biological process would be necessary to bring this about?

A GENETICS PROBLEM (NEUROSPORA) – work in pairs

You should know what all the terms in bold type mean.

A. The ascomycete Neurospora is a haploid, coenocytic fungus which produces asexual conidiospores on aerial hyphae growing out of the coenocytic mycelium.

In this strain of Neurospora each conidiospore carries a single nucleus.

A series of mutant strains which fail to grow in the absence of arginine were isolated over a period of months. They were all produced in strains with the same mating type.

An experiment was set up to determine how many genes these arginine-requiring mutants represent. The mutant strains were taken in pairs and the two strains allowed to grow together in the presence of arginine and to fuse forming a single hypha containing nuclei from each parent strain. A hypha which contains a mixture of genetically different nuclei is called a heterokaryon. The genetically different nuclei in a heterokaryon do not fuse but remain as separate haploid nuclei within a single multinucleate hypha. Heterokaryons were tested for their ability to grow without arginine; the following results were obtained.

(+ = growth, - = no growth)

Strain Strain 1 2 3 4 5 6 7 8 1 - + + + + + + - 2 - + + + - - + 3 - + - + + + 4 - + + + + 5 - + + + 6 - - + 7 - + 8 - 49

QUESTIONS

Al. How many genes do the eight mutants represent? Which mutants are alleles at the same gene locus?

A2. What information are you given above which allows you to conclude that the results in the table come about from complementation and not recombination?

A3. How might you show unambiguously that the growth obtained when certain pairs of mutants form a heterokaryon does not involve genetic exchanges (i.e. recombination) between them?

B. Four possible intermediates (A-D) in the biosynthesis of arginine have been obtained in a pure, stable form and used as supplements to see if they would support growth of the mutants.

The results were as follows:-

Medium supplemented with substance: A B C D Arginine Strain 4 - - - - + 5 - + - - + 6 - + + - + 8 - + + + + (+ indicates growth, - indicates no growth)

QUESTIONS

See if you can use these data to: B1. Deduce the order of these compounds in the biosynthetic pathway of arginine and B2. Determine which mutants block which steps in the conversion of these intermediates into arginine?

C. Whilst conidiospores are produced by asexual reproduction (see introduction), ascospores are generated from sexual reproduction. Thus if two different mating types are allowed to grow together, they will fuse to form a diploid zygote. Meiosis of this zygote then gives rise to haploid ascospores. Mutants 2, 6 and 7 were taken through crosses to change their mating types so they could be crossed together. A million ascospores from each cross shown below were plated on minimal medium to select rare recombinants which are arginine independent. The number of prototrophs from each cross are given:-

Cross Number of Prototrophs Number of Recombinants 2x6 3 2x7 20 6x7 22

QUESTIONS

C1. Complete the third column in the table above; do the arginine independent recombinants represent all the products of these recombination events? C2. Calculate the recombination frequency and then construct a map for the arginine region of the chromosome and show map distances between mutations.

D. Finally an arginine dependent mutant strain X, which fails to complement mutants 2, 6 and 7, was crossed to each strain but no arginine independent recombinant ascospores were recovered. Strain X clearly has a different class of genetic change from the other mutant strains.

D1. Suggest what sort of mutation might give rise to strain X.

HOMEWORK EXERCISES:

There are two exercises that you should do in your own time before the next practical. Please remember to bring your answers/results with you to Practical 4.

Homework Exercise 1:

Lac Operon– Learn In the course section on Learn you will find photographs of E. coli grown on L-broth agar plates containing either lactose or glucose as a carbon source. Three strains of E. coli have been used to investigate the activity of -galactosidase. The presence of active enzyme is indicated by blue colonies, the absence of active enzyme by white colonies. Full instructions and descriptions can be found on Learn and you should answer the questions posed. In the next practical there will be plates available for you to look at and you will have the opportunity to ask the demonstrators any questions you may have on this exercise.

Homework Exercise 2:

Estimating the DNA Fragment Sizes from Restriction Enzyme Digestion- Data provided below

This exercise will prepare you for the next two practicals. You will work out the estimate of the fragment sizes from the data below and then in the next practical you will use your estimates to build a restriction map of the plasmid.

You are provided with a diagram of an electrophoresis gel showing the DNA fragment patterns produced by cleaving a small, circular bacterial plasmid DNA with various enzymes, either singly or in pairs. Lanes 1 and 8 show patterns produced by size standards. The information is sufficient for you to construct a restriction map of the plasmid for these enzymes (which you will do in Practical 4).

Instructions for Measuring the Size of DNA Fragments

DNA fragments are separated according to size by electrophoresis in either agarose or polyacrylamide gels. Sizes up to about 20 kb pairs may be separated in agarose gels, while smaller fragments in the range 10-100 bp are only well resolved in polyacrylamide gels. It is usual to include a set of standard marker DNA fragments of known sizes in 51

separate lanes. A standard curve (which is S-shaped but is approximately linear over its central portion) is constructed by plotting the log of fragment size against mobility. The mobility of individual fragments are measured as accurately as possible and their sizes estimated from the standard curve by interpolation.

You are advised to use the following strategy:

1. From the diagram of the gel in your coursebook, measure the distance from the well to each band (choose the front of the well and the front of the band) for each marker standard.

2. Plot the graph of fragment size on the log scale versus distance migrated using the three cycle log paper provided in this book. Draw the best fit straight line through the points corresponding to the seven smallest fragments (but plot all eight size markers).

3. Measure the distance migrated from the well for all fragments of plasmid DNA and estimate their sizes from your graph.

Note: Fragment sizes that you obtain will be approximate. Don't worry if there are discrepancies of a few hundred base pairs when you come to calculate how they all fit together to give your circular plasmid map in Practical 4.

Bring your standard curve and size estimates with you to Practical 4 and please also bring a ruler.

M M a MARKER SIZES ARE IN KILOBASES a r r k Sal1 Sal1 Xho1 k e e Sal1 Xho1 Xho1 Pvu2 Pvu2 Pvu2 r r

Wells s s

23.6 23.6

9.64 9.64

6.64 6.64

4.34 4.34

2.26 2.26

1.98 1.98

0.95 0.95

0.63 0.63

PRACTICAL 4

MAPPING RESTRICTION SITES IN PLASMID DNA –PART 3

Resolution of DNA Fragments by Gel Electrophoresis

The plasmid DNA, which you prepared and digested with restriction enzymes last time, has been returned to you and you now need to subject the treated DNA to gel electrophoresis to separate the DNA fragments. To do this follow the procedure outlined below.

Procedure:

1. Pipette 4 l of Loading Buffer into each of the tubes containing your restriction enzyme digests and control.

2. Vortex all tubes briefly to mix (2 sec).

3. Spin in microfuge for 10 sec.

Note: the loading buffer contains a dye to track the progress of the electrophoresis and will also stop the enzymatic digestion. It also contains a dense polysaccharide (Ficoll) which helps the sample sink to the bottom of the loading well.

4. Using a Gilson P20 pipette, load 20 µl of your samples onto gel. Your demonstrator will remind you how to do this by loading molecular weight markers into the first well.

The order of samples for your group should be as follows:

(a) Marker (b) Undigested control plasmid A (c) Plasmid A cut with EcoRI (d) Plasmid A cut with HindIII (e) Plasmid A cut with HindIII / EcoRI (f) Undigested control plasmid B (g) Plasmid B cut with EcoRI (h) Plasmid B cut with HindIII (i) Plasmid B cut with HindIII / EcoRI

5. Note the number of the gel and also the order in which the samples are loaded, including the molecular weight markers.

6. When all the samples are loaded, the demonstrator/technician will switch on the power and the gel will be run until the tracking dye is near the bottom of the gel.

7. The gel will be carefully removed from the electrophoresis chamber and place in the stain solution, by the laboratory technician who will stain it for 20 minutes.

8. The gel will be photographed and the data will be available for analysis in the next practical

YEAST EXPERIMENT --- PART 3

The test plates that you set up last time are returned to you for recording the results. Examine the plates and determine which strains complement one another. Use this information to decide which histidine strains have mutations in the same gene and which are different. Write out the complete genotypes of the strains you tested.

INTRAGENIC RECOMBINATION WITH rII MUTANTS OF BACTERIOPHAGE T4

Rapid lysis mutants of bacteriophage (phage) T4 produce large plaques on the B strain of E. Coli but fail to grow on the K12 () strain. Mutation in any one of 3 gene loci— rI, rII or rIII — will produce the rapid-lysis phenotype.You will use phage T4 rII- mutants to show that two mutations which fail to complement (i.e. mutations in the same gene) can still recombine to give rII+ wild type phage particles. rII- mutant strains were isolated by infecting a lawn of E. coli B at a low multiplicity of infection (MOI). At low MOI only single phage particles should infect cells. After replication within the cell, phages cause lysis (bursting of the cell membrane) which kills the cell and release more phage of the same genotype as the original infection starting further rounds of infection and lysis in adjacent host cells. Eventually an original single bacterial cell infected by a single phage is detected as a single plaque (a cleared area) in the lawn of bacteria. When rII+ and rII- phages are tested on the two bacterial strains E. coli B and E. coli K12 () characteristic different plaque morphologies are found. Thus:

Host Bacterial Strain

E. coli B E. coli K12 ()

Phage

rII+ small plaques plaques

rII- large plaques no plaques

First objective. To show that you have genuine rII- mutants by testing their growth on E. coli B and E. coli K12 (). What do you expect to see if you do have genuine rII- mutants?

Second objective. To show that the mutations fail to complement each other. A mixed suspension of phages infects E. coli K12 () at high MOI. If you have two genuine rII- mutant phages neither will be able to grow and no plaques will be formed. However if the phages are mutant in two different genes (e.g. rI and rII) they will complement each other and lysis (i.e. plaque formation) is possible.

Third objective. To test for recombination between two non-complementing rII- mutants to give rII+ phages. T4 phages will only recombine during DNA replication and so each mutant must be present within the same E. coli B cell, achieved by a mixed infection at high MOI. If recombination has taken place in E. coli B rII+ phages will be released, and these wild-type phages may then infect and lyse E. coli K12 (). 56

Materials supplied:  Two phage T4 strains called W and Y  Three agar plates  Three tubes of molten top agar at 48C  One tube of E. coli B (see step 2)  One tube of E. coli K12 () (see step 2)  Pipette suitable for measuring 50 l  Pipette suitable for measuring 10 l  Sterile pipette tips.

You will work in groups of four for this experiment.

METHOD

1. Label the bottom of one agar plate ‘E. coli B’, and the other two ‘E. coli K12 ()’.

2. Bijou bottles containing aliquots of E. coli B and E. coli K12 (λ) are in the 37oC incubator. You should take one aliquot of each strain just before you are ready to use the cells, remembering to gently mix the bottles before use.

3. Take one tube of top agar, add 0.2ml of E. coli B, vortex briefly, pour onto correctly marked plate, roll plate to spread top agar over whole surface of the plate, leave to set.

4. Take one tube of top agar, add 0.2ml of E. coli K12 (), vortex briefly, pour onto correctly marked plate, roll plate to spread top agar over whole surface of the plate, leave to set.

5. Repeat step 4 for second plate marked E. coli K12 (). These plates when incubated overnight will grow what is called a "lawn" of bacteria over the whole surface of the plate.

6. While top agar is cooling label and set up three Eppendorf (microfuge) tubes as follows: i) 0.1 ml T4 mutant W alone. ii) 0.1 ml T4 mutant Y alone. iii) A mixture of T4 mutants W and Y, 50 l of each phage suspension is sufficient.

7. Spot phage from each tube onto a plate you have overlaid with each bacterial strain as follows  Mark the E. coli B plate and one of the E. coli K12 () plates as indicated in the diagram.  Using a clean, sterile pipette tip spot 10 l of phage suspension from tube i) onto a well separated marked area of each plate.  Repeat for tubes ii) and iii) using fresh clean, sterile tips each time.  You will have two plates, one E. coli B and one E. coli K12 (), which should look this. E. coli B E. coli K12 ()

8. Prepare your second E. coli K12 () plate as follows:-

 Add 50 l of E. coli B cells to each of the three tubes of phage that you prepared in step 6 and incubate them for 30 minutes in a 37oC water bath, using a floating rack.  Mark the plate as above and remember to label it so you can distinguish it from the other E. coli K12 () plate used above (7.).  Using a clean, sterile pipette tip spot 10 l of phage suspension from tube i) onto a well separated marked area of the plate.  Repeat for tubes ii) and iii) using clean, sterile tips each time.  You will now have a third plate, E. coli K12 (), which should look like the first two.

9. Before you leave - Make sure your group ID is on the bottom of each plate. Place the plates in the wooden rack provided and leave them for incubation at 37C.

GENOMIC PCR EXPERIMENT -PART 4

ANALYSIS OF REPETITIVE DNA AT THE HUMAN LOCUS D1S80– determining the number of repeats present in your alleles at the D1S80 locus

From the photo of the PCR results from your group, draw a standard curve using the semi- log graph paper provided and work out the sizes of the PCR bands.

Now determine the number of repeats that are present in the alleles at the D1S80 locus.Please add your results to the class data sheet provided.

QUESTIONS: 1. Why are two bands commonly seen? 2. Why are the bands often different in size? 3. Why are there sometimes faint ‘extra’ bands? 4. Why do you think there are no bands in some tracks? 5. Which are the most common allele sizes? 6. What is the frequency of these alleles within the class?

LAC OPERON EXERCISE – Learn – Homework exercise 1; PART 2 Agar plates containing either lactose or glucose as the carbon source will be available for you look at. This supports the exercise on Learn that you were asked to complete for today’s practical. Please check your results from this exercise with your demonstrator.

A Set of Restriction Enzyme Digestion Data –Homework exercise 2; PART 2

You will now use the fragment sizes you were asked to calculate from the gel picture in the coursebook as homework exercise 2 in Practical 3.

Construct a circular restriction map of the plasmid by considering the data in the following order:  the SalI and XhoI single and double digests  the PvuII single digest  the PvuII/SalI and PvuII/XhoI double digests NB. You may find it more convenient to draw the circular restriction map as a straight line with the same restriction site at both ends – try both approaches. 58

PRACTICAL 5

INTRAGENIC RECOMBINATION WITH rII MUTANTS OF BACTERIOPHAGE T4 – PART 2

You will have 3 plates (one E. coli B and two E. coli K12()) from the previous practical. Analyse the results of your mixed infection of E. coli B cells with the two T4 mutants.

QUESTIONS:

1. Why did you add E. coli B cells to the phage before spotting onto the second E. coli K12 () plate? 2. How can you distinguish between recombination and complementation in this system?

MAPPING RESTRICTION SITES IN PLASMID DNA – PART 4

Measuring the Size of DNA Fragments

From the photograph of your gel from the previous practical, and using the same strategy as for the gel picture in the course book, measure the distance from the well to each band, plot the graph of log fragment size versus distance migrated and draw the best fit straight line through the points corresponding to the marker fragments.

Now use the graph to estimate the sizes for all fragments of your digested plasmid DNA.

Note: As before, fragment sizes that you obtain will be approximate. Don't worry if there are discrepancies of a few hundred base pairs when you come to calculate how they all fit together to give your circular plasmid maps.

Finally, construct a circular map of each of the plasmid DNA molecules from strain A and B and compare them.

QUESTIONS:

1. How many restriction sites are there for EcoRI in i) plasmid A and ii) plasmid B? 2. How many restrictions sites are there for HindIII in i) plasmid A and ii) plasmid B? 3. What is the approximate size of the inserted PstI fragment of the human α-globin gene in recombinant plasmids A and B? 4. Why are the maps for plasmids A and B different?

MAPPING GENES IN HUMAN CHROMOSOMES Human family pedigrees

Pedigrees of human families showing particular phenotypes have provided invaluable genetic data on inheritance, segregation and linkage of some human genes. Pedigrees are normally drawn as in the figure below with one generation (Roman numerals) per line and individuals numbered (Arabic numerals) from the left. Individuals are denoted by single symbols which indicate sex and phenotype. Spouses are joined by a single short horizontal line and their offspring are grouped together, oldest to the left, youngest to the right, joined by lines. Often other information (deduced by genotype, other relevant genetic information (blood group, rhesus factor) and age) is added (normally written below or adjacent to the reference symbol).

Symbols Phenotype Male Female

Unaffected

Affected

Dead

Unaffected Carrier

Generation I 1 2

II 1 2 3 4 5 6 7 8 9 10 11 12 13

III 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

IV 1 2 3 4 5 6 7 8 9

This preliminary pedigree includes information about affected and unaffected individuals but generation IV requires further analysis and completion. Using your knowledge of Mendelian genetics answer the following questions related to this pedigree.

1. What is the mode of inheritance of this trait? Give reasons for your answer. 2. What can you say about individual I.2? 3. What can you say about individuals IV.1, IV.6 and IV.9? 4. Do you now consider that a re-write of this pedigree is necessary? If so, why?

Analysis of Human Pedigree Genetic Data

1. Establish the pattern of inheritance for each phenotype in turn. (Is it autosomal, sex-linked or sex limited? Is it dominant, co-dominant or recessive?) This will require counting affected and unaffected males and females separately, and inspecting the progeny of affected individuals.

Autosomal linkage Males and females equally affected

Sex-linkage X-linkage Affected males never pass an X-linked trait to sons, X- linked recessive alleles are expressed in all male carriers, females may be unaffected heterozygous carriers for recessive X-linked traits

Y-linkage Males only, affected males always pass trait to sons, females are never affected and can never pass on trait Sex-limitation Male-sex-limited Males only affected; if dominant, affected males may (autosomal) pass trait to sons and may have daughters who are unaffected carriers.

Female sex-limited Females only affected; if dominant, affected females (autosomal) may pass trait to daughters and have sons who are unaffected carriers.

Dominant phenotype All carriers affected

Co-dominant phenotype Heterozygotes express both phenotypes

Recessive phenotype Only homozygotes affected; Alleles of an X-linked gene will be expressed in males because they only have one X chromosome and thus only one allele of the gene.

2. Linkage is most easily established when two loci are so close together on the same chromosome that recombination is infrequent and linked genes almost always co- segregate. The further apart two loci are the more difficult it is to be sure of linkage. When two loci are separated by 50 map units or more they will not show linkage but assort independently and no matter how many gametes are counted the loci will always appear to be unlinked (independently inherited). Therefore the further apart two loci are the greater the number of gametes (progeny) which have to be screened before you can be sure of linkage. Human pedigrees are not ideal for linkage analysis because family sizes are small compared with the number of progeny needed to establish linkage. However it is permissible, in favourable circumstances, to pool data from several different families. Clearly the more closely linked two loci are the easier it is to prove linkage by pedigree studies.

3. To establish linkage between loci consider pairs of loci in turn. You have to show that the loci are co-segregating (linked) not independently assorting (unlinked).

4. Consider each mating in turn and deduce the genotypes of parents from their phenotypes taken together with the phenotypes of their progeny. Find informative

matings which will be between the double heterozygote, such as AaBb, and the double recessive homozygote, aabb. If loci A and B are unlinked there should be 25% Aabb, 25% AaBb, 25% aabb and 25% aaBb progeny. If they are linked the 1:1:1:1 ratio will be distorted depending on the coupling phase (that is which particular alleles are together on the same chromosome). In such a mating, if the loci are linked, the products of all recombination events which took place in the double heterozygous parent will be detectable as the least common class of progeny. Thus if A is coupled to B and a is coupled to b:-

Parental Gametes Gametes Coupling Genotype NR R Phase AaBb AB ab Ab aB AB, ab X aabb ab AaBb aabb Aabb aaBb

With opposite coupling the table will be:- Parental Gametes Gametes Repulsion Genotype NR R Phase AaBb Ab aB AB ab Ab, aB X aabb ab Aabb aaBb AaBb aabb

In both cases all non-recombinants (NR) are distinguishable from all recombinants (R). If the loci are independently assorting the number of "recombinants" should equal the number of non-recombinants. But if the loci are linked the number of recombinants will be much less than the number of non-recombinants and the ratio of recombinants to total progeny will be an estimate of the recombination fraction (or frequency). With small numbers of progeny and loci which are well separated on the genetic map it may be difficult to decide whether the data indicate linkage. There are statistical techniques for helping with linkage analysis but they are beyond the scope of this course.

For each informative mating make a table of the four progeny classes with the number of each. From these numbers you should calculate an estimate of the recombination fraction or frequency. Pool data for different families in the pedigree for a better estimate and decide if the loci are linked.

5. Co-dominant alleles are easier to work with because phenotypes directly reflect genotypes and because coupling phase can be established more easily. For instance the Adenylate Kinase locus has two alleles which are both expressed in heterozygotes. The human blood group ABO antigens are controlled by a locus with three alternative alleles A, B and O. A and B are co-dominant but O is recessive and detectable only as absence of A and B. AB heterozygotes are detectable but not AO or BO, which have phenotypes like AA and BB respectively. If both loci have co- dominant alleles then a greater number of matings are potentially informative. Again a double heterozygote AB12 is required but may be mated to any double homozygote (AA11, AA22, BB11, BB22) providing coupling is known. It is not possible to use matings between a double heterozygote and one of the single heterozygotes (AA12, BB12, AB11, AB22) since only 50% of recombinants are detectable. 62

HOW TO PROCEED WITH A PEDIGREE ANALYSIS

 Consider ONE pair of loci and each mating in turn.  For each mating write deduced parental genotypes.  For each parent write genotypes of all possible gametes.  Make a table of gametes from one parent against gametes from the other parent.  Mark each offspring on the table according to PRESUMED genotype.  Is the genotype ratio among progeny near 1:1:1:1 (no linkage) or 1:1 (linkage)?  Deduce which alleles are coupled (inherited together).  Deduce recombinant class(es) (lowest frequency genotype(s)).  Calculate a recombination fraction for each informative family separately.  Calculate a total recombination fraction for all informative families.

ANSWER THE FOLLOWING QUESTIONS

1. For your chosen pair of loci calculate a recombination fraction. The recombination fraction (or frequency) is the number of recombinants divided by the total number of progeny from informative crosses. An informative cross is one between individuals in which all the products of recombination lead to detectable recombinant progeny. Thus if A and B are linked, an informative cross would be between AaBb and aabb but not between AaBb and AABB, AABb or AaBB.

2. Get data on the other two pairs of loci from another group or demonstrator.

3. Draw a map of this region of the human genome.

4. What is the mode of transmission of Nail-Patella Syndrome? 5. How could you improve the reliability of conclusions from these data?

6. Why are X-linked genes easier to work with than autosomal-linked genes?

7. Why are alleles that give a dominant phenotype easier to work with than those that give a recessive phenotype?

8. Suggest ways in which restriction enzymes might help pedigree analysis.

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