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Welcome to Biol 342
Molecular Biotechnology 1
Dr. Michael D.J. Lynch

Biology 342
Molecular Biotechnology 1

Instructor: Dr. Michael D.J. Lynch
Room: B2 - 249D e-mail: mdjlynch@uwaterloo.ca office hours: Thursdays 1:00 – 2:30 pm
If you need to speak with me outside scheduled lecture time, please contact me via email to make an appointment – that way I can be sure to set aside time for you.
Prerequisites: Biol 130, 239, 240, 309. Biol 241 recommended
Required textbook: Glick & Pasternak Molecular Biotechnology 4th edition, 2010. ASM Press. Available from UWaterloo Bookstore.
2 copies on reserve at Davis library. Students find this textbook very useful, and I refer to it often for lectures. A worthwhile purchase. This text is also used in Biol 432.

LEARN







lecture notes (slides in .pdf)
Podcasts (screencasts) of lectures course info, important dates tutorial information practice exams announcements Use your Quest/UWdir ID and password

Accessing the podcasts……..
Check that you are using a LEARN-approved browser!

Goals for this course:

Understand the fundamentals of molecular biotechnology, primarily in the context of the methods that are employed in the field

Develop skills in the designing of molecular approaches to biotechnology ●
Develop critical thinking skills

Effectively communicate concepts learned

Assigned readings and student notes: Readings from the text will be assigned in lecture notes on the course website (accessed through LEARN). Please complete readings before each relevant lecture. I will make available sets of “notes” from which all lecture material will be based, that you can download for printing (again on
LEARN). I recommend that you print these and bring them to lecture to make additional notes from. They are designed to facilitate note-taking, and are not necessarily a complete set of lecture notes.
This course description/outline is also available at this site
(see Syllabus).

General Sequence of Lecture Topics:









Introduction, History, Review of basic gene expression
Review of cloning procedures and vectors, libraries
Synthetic DNA, PCR, DNA sequencing methods
Reverse translation, codon usage
Recombinant protein expression - prokaryotes
Molecular diagnostics

monoclonal antibodies, ELISA, molecular beacons, genotyping techniques
Recombinant protein expression - yeast, insect, mammalian

Tutorial topics
(sometimes these change slightly, check LEARN for updates)
1. Gene structure review, locating ORFs, reverse translation, operons
2. Advantages of double digests, selection, cloning using pBR322 and pUC, designing a cloning vector 3. Expression of cloned genes, calculation of colony # for a genomic library, vector choices, functional complementation, drawing products of PCR
4. Designing PCR primers for cloning PCR products, searching for unknown genes given a known protein, pL and pT7 promoters, tagged fusion proteins, high-throughput vs Sanger sequencing. 5. Designing E. coli expression vectors, cloning in-frame into pGEX vectors
6. Folding of recombinant proteins: cloning cDNA in pGEX; finding autoantibodies in ulcerative colitis using phage display
7. Integrating multiple gene copies into the chromosome
8. Monoclonal antibodies, designing a test using molecular beacons
9. Designing dip-stick tests for HIV and pesticides

Evaluation
Midterm Exam

25% Written answer

Tutorials

15% Submitted group solutions

Final Exam

55% T/F, short answer, written

Assignment

5% Post Midterm

Tuesday, June 23rd
(in class)

TBA
Tuesday, July 21st

All exams and tutorials must be normally completed in order to qualify for a pass in this course

TUTORIALS: Attendance for the tutorial sessions is mandatory. ●



An absence without a valid medical certificate, will result in an automatic zero on the missed tutorial. Make-up tutorials will not be allowed.
If you completely miss a tutorial, and your absence is deemed legitimate by myself or your TA, then that percentage will be equally distributed among your other tutorials, so as not to result in a penalty.

Tutorial sessions: This is group work (groups are preassigned;~ 4 per group). At the end of each tutorial your group will submit completed answers to the tutorial problems. This is a cooperative learning activity, so notes and textbook are a must for these application-type questions. You must attend the tutorial session to which you are assigned. Expect questions on the exams relating to the tutorial material (in other words, attend all tutorials!).
One TA will be assigned to each tutorial section. They will be able to assist you with your tutorial assignments and will grade these.
In the first tutorial session (May 15), they will give you details on how best to contact them if you need help outside tutorial time. There is NO tutorial June 19 (preceding midterm). last tutorial is July 17 (see results during office hours)
Your TAs is James Campbell

GROUP CONTRACT FOR BIOL 3
42

Posted on LEARN for viewing You’ll be required to sign this in Tutorial 1
I will post the tutorial topics ahead of time on
LEARN so you can prepare for the tutorials

Be haviours e cte of e xpe d ach g roup m m r e be
Improving the quality of the tutorial experience involves engaging in collaborative discourse where constructive exchange of each other’s ideas offers a means for more deeply learning the science required to answer questions.
This can only happen in a group setting where ALL members participate.
Be pre pare d:
1. All group members will be punctual and everyone should be there and ready. Any group member who is 10 min or more late, may not join the group and must complete the tutorial assignment on their own.
2. All group members will remain until (a) all tasks are completed, or (b) there is unanimous adjournment.
Ge involve t d;
1. All group members will come prepared by (a) checking the LEARN course site for the tutorial topics, and (b) reviewing the relevant material (notes, text, etc.).
2. Roles that group members agree to undertake must be adhered to (e.g., photocopying, supplying a textbook etc.) 3. Each group member has the right to point out whether any of these responsibilities are not being fulfilled.
4. Each group member is responsible for signing his or her own name and ID number on both the attendance list and the assignment sheet. Failure to do this will result in zero for that tutorial for that member.
Be re ctful: spe 1. The group will actively seek a consensus of opinion based on the opinions of every member.
2. Each member will take turns listening as well as talking, and active listening will be a strategy for all group discussions. 3. Sexist or racist or other discriminatory remarks and aggressive or dominating behaviour are NOT acceptable.
If you are feeling frustrated, take a deep breath before you speak. It is incumbent upon all group members to gently but firmly point out behaviours that cross or even get near this line.
4. Disagreements will be handled with courtesy, respect, and diplomacy within the group at all times. If there is a serious disagreement within the group that cannot be resolved by the group, the TA must be informed.
5. Each member of the group shall contribute to the task. Marks for completed tutorials will be distributed evenly amongst the present group members.
We them m rs of the g e be roup ag eto the e ctations outline in this contract. re xpe d Sig d on this date ______________________________ ne :
1. Me mbe r

Print name

Sig d ne 2. Me mbe r

Print name

Sig d ne 3. Me mbe r

Print name

Sig d ne 4. Me mbe r

Print name

Sig d ne (Adapted fromEnglish 409 A/B 1991 CourseNote by D. Goodwin and Le s arning in Te s a Stude Guide am : nt by GrahamGibbs, for the TRACE workshop on Preparing Students for Group Work, February 16, 2000)

Assignment 5%
Assigned after the midterm
Can hand it in early for feedback and re-submission
Last day for early submission/feedback will be Tuesday, July
14th. I’ll hand back Friday, July 17th to make final submission date of Tuesday, July 21st.
~ 3-4 weeks to complete

Attendance and Class Policy:
To give yourself the best opportunity to do well in this course, attendance for the lecture session is highly recommended. The midterm and final exam contain material selected from both the lecture and tutorial portions of the course. Lectures and tutorials are designed to emphasize particular aspects of the course. As well, lectures present additional visual and oral material that is designed to stimulate thought and interest in the field of biotechnology.

EXAMINATIONS: All tests must normally be written in order to qualify for a pass in this course. Failure of both the midterm and final exam will result in a failing overall grade. In this case, the final exam grade will become the final grade.
Student travel plans are not considered acceptable grounds for granting an alternative examination time.
See:
http://www.registrar.uwaterloo.ca/exams/finalexams.html#info and http://www.adm.uwaterloo.ca/inforeg/exams/ExamRegs.pdf

A VALID medical certificate (see next) must be supplied in order to qualify for a weight transfer to the final exam, or a tutorial % transfer, or to qualify for a make-up exam.
Make-up midterms will not normally be allowed because of the large size of the class. If you miss the midterm (due to a legitimate reason as deemed by the instructor), then that weight will be transferred to the final exam. In this case, you must pass the 80% final exam to pass the course.
The make-up final exam for the Fall semester will normally be scheduled for February reading week. For the Spring semester, a make-up exam will be scheduled for the end of frosh week or the beginning of Fall classes.
It is your responsibility to contact me before this to get the day and time. A valid medical certificate (available at info.uwaterloo.ca/infoheal/_StudentMedicalClinic/VIF.html) must specifically state examination by a physician on the day of the illness and must not be after-the-fact. Compassionate leave (e.g., death in the immediate family) will be granted by the instructor/lecturer on a case-by-case basis and will normally require validation by documentation. Please make contact as soon as possible in this event so judgment can be made before the examination. Note: for purposes of what constitutes a doctor or physician, that person must be registered in the College of Physicians and Surgeons of Ontario. It is NOT an automatic right of students to be granted deferrals! Please see https://uwaterloo.ca/science/current-undergraduate-students/conse quences-associated-submission-verification-illnessform
For further information regarding missed tests/exams, please refer to: http://www.registrar.uwaterloo.ca/exams/finalexams.html#info www.registrar.uwaterloo.ca/exams/ExamRegs.pdf http://biology.uwaterloo.ca/undergraduate/regulations-for-academic-discipline https://uwaterloo.ca/science/current-undergraduate-students/consequences-associatedsubmission-verification-illness-form

Drop dates - to watch!
May 22
June 19
July 10
After that

No penalty and tuition refund
WD and 50% refund
WD and no refund
WF = 32%

Academic Integrity:
In order to maintain a culture of academic integrity, members of the University of
Waterloo community are expected to promote honesty, trust, fairness, respect and responsibility. Avoidance of academic offences:
Discipline: Students are expected to know what constitutes academic integrity, to avoid committing academic offenses, and to take responsibility for their actions. Students who are unsure whether an action constitutes an offense, or who need help in learning how to avoid offenses (e.g., plagiarism, cheating) or about ‘rules’ for group work/collaboration should seek guidance from the course instructor, academic advisor, or the Associate
Dean of Science for Undergraduate Studies. For information on categories of offenses and types of penalties, students should refer to Policy #71, Student Discipline. For information on typical penalties, students should check
Guidelines for the Assessment of Penalties.

Guidelines for Assessment of Penalties, UW Faculty of Science
DO NOT:

•Use inappropriate citations on written assignments
•Share assignment answers (avoid excessive collaboration)
•Submit same material for more than one assignment/course

Any of the above activities is deemed an academic offense and will be penalized as follows:
•The assignment in question will be given a mark of zero.
•A letter, signed by the Instructor and student, outlining the offense and grade change will be forwarded to the Associate Dean of
Undergraduate Studies, Faculty of Science who will decide if additional penalties need to be be applied.
•Copies of this letter will be placed in the student’s file.
Second infractions or more serious offenses (e.g. fraudulent use of medical certificates or any cheating on exams or tests) will incur further penalties and be handled on an individual basis by the Associate Dean of
Undergraduate Studies, Faculty of Science. For details refer to
“Regulations for Academic Discipline”.

Academic Offenses
Any attempt on the part of a student to gain an unfair advantage may be construed as academic misconduct, irrespective of whether any benefit was gained by the student(s) concerned. UW academic regulations

Unauthorized co-operation or collaboration (excessive collaboration) This is a form of plagiarism that occurs when someone writes sentences, or constructs paragraphs for you. This also applies to art, graphs, diagrams or figures, data, and calculations. Unintentional plagiarism (e.g., I was just getting help; I ran out of time; we wrote the sentences together) is still plagiarism. It is not the instructor’s job (and it is not possible), to uncover a student’s motives for plagiarizing. It is what it is.
Students should treat their academic work as their own property. It is the responsibility of students to protect their own work. Students should ensure that electronic copies of their work are stored securely and cannot be copied or stolen by another person; for example, in computer laboratories. Allowing someone to represent your work as their own is also academic misconduct.

Examples of plagiarism and excessive collaboration













working with another person on an individual assignment/test having someone else complete all or part of your assignment for you having someone rewrite your assignment for you obtaining, or attempting to obtain, assistance with an individual assignment, for example, by using an essay-writing service obtaining, or attempting to obtain, access to assignment/test materials (eg., examination/quiz/ i-clicker questions) including questions/answers/marking schemes during, prior to, or after the assignment/test without explicit permission from an instructor or TA. This includes obtaining access to screen shots, cell phone/i-pad pictures etc., of above materials. copying, communicating, or attempting to copy or communicate, assignment/test materials (eg., examination/quiz i-clicker questions) including questions/answers/marking schemes during, prior to, or after the assignment/test without explicit permission from an instructor or TA. This includes taking and passing along screen shots, cell phone/i-pad pictures etc., of above materials. copying, or attempting to copy, the work of another student or instructor and submitting it as your own. submitting work produced in collusion with another person(s) for an assignment that is to be assessed on individual work. inclusion of unauthorized members in student teams when conducting group work assignments. lending work that has been submitted for assessment to another student.

It is the student’s responsibility to ensure they have understood the definition of academic offences. See Office of Academic Integrity ( http://www.lib.uwaterloo.ca/ait/misconduct.html) for more information and seek clarification from your course instructor if you are still not clear on what constitutes an academic offence.
Note that if the extent of the collaboration allowed for an assignment or lab is not stated explicitly in the syllabus, students must assume that absolutely no collaboration is allowed.
Plagiarism is unethical because:







It diminishes the value of another’s original work
It gives an unfair advantage over the student who does his or her own work
It undermines student learning, respect for the learning process, and the value of academic achievement
Unchecked plagiarism devalues the worth of the degree, the reputation and credibility of students, faculty, and the institute itself

Student grievances
Students, who believe that a decision affecting some aspect of their university life has been unfair or unreasonable, may have grounds for initiating a grievance. Students should read
Policy #70, Student Petitions and Grievances, Section 4. When in doubt, students must contact the department’s/school’s administrative assistant who will provide further assistance. http://www.adm.uwaterloo.ca/infosec/Policies/policy70.htm Appeals
A decision or penalty imposed under Policy 33 (Ethical
Behavior), Policy #70 (Student Petitions and Grievances) or
Policy #71 (Student Discipline) may be appealed, if there is a ground. Students, who believe they have a ground for an appeal, should refer to Policy #72 (Student Appeals).

Students with disabilities
AccessAbility Services (AAS), located in Needles Hall, Room
1132, collaborates with all academic departments to arrange appropriate accommodations for students with disabilities without compromising the academic integrity of the curriculum. If students require academic accommodations to lessen the impact of their disability, they should register with the AAS at the beginning of each academic term.

If you are planning on pursing a career in a field that uses
Molecular biology, then be sure to take BIOL 335L to get technical skills!

Chapters 1 - 2 text
1) What is Molecular Biotechnology?
2) Review of basic gene expression prokaryotes vs eukaryotes

Biotechnology

Biotechnology

Biotechnology

Biotechnology

Biotechnology
The science of using organisms to produce a product for a commercial use. Commonly takes advantage of an organism’s metabolic system.
e.g., cleaning up of oil spills uses cultures of specialized microorganisms (e.g. Pseudomonas species) that naturally catabolize complex hydrocarbons
Wine making-uses fermentation by yeast (Saccharomyces ellipsoideus), which produce ethanol as a natural product of metabolism of sugars

Bioplastics






Mirel (polyhydroxybutyrate, PHB), a clear, biodegradable, plant-based plastic

characteristics similar to polypropylene
Produced by fermentation using a proprietary bacterial strain, producing PHB polymer from corn sugar (i.e., brewing plastic)
Metabolix produces many types of biopolymers also based on polyhydroxyalkanoate polymers (PHAs)

you have all used these bioplastic products

http://www.metabolix.com/

Molecular Biotechnology

Molecular Biotechnology

Molecular Biotechnology

Molecular Biotechnology

Molecular Biotechnology

Molecular Biotechnology

Molecular Biotechnology late 70s, early 80s
● manipulation of DNA to enhance a natural product (protein) or add a new one


e.g.,
● introduction of two directed mutations in the gene coding for xylose/glucose isomerase from Clostridium thermosulfurogenes resulted in a large increase in specificity for glucose which it then converts to fructose
● important for the food industry (syrup)


insertion of human insulin gene into E. coli for large-scale production milestone pg 4-9 text
Cohen, Chang and Boyer’s work - 1973
● first demonstration of gene cloning using plasmids as vectors
● others were quick to follow up
● lead to design of vectors and hosts specifically for this purpose
● also lead to a voluntary moratorium on gene cloning where the genes to be cloned were thought to be potentially dangerous
● e.g., cloning of genes coding for bacterial toxins

most institutions currently have committees which approve/reject proposed genetic modification of organisms however, they are not all uniformly rigorous and
“unforseen” results sometimes occur…………….

Australia’s mouse problem

1998
Cloned a gene for mouse sperm receptor into a relatively harmless mouse virus (mouse pox) in order to get female mice to produce an immune response to the receptor so that fertilization could not occur. didn’t get much of an immune response (low antibody production) turns out that this virus induces a cell-mediated immune response vs an antibody response in mice, so the researchers added another mouse gene to the virus (IL-4)
IL-4 pushes the immune system towards an antibody response, therefore the researchers were hoping to increase the level of antibody to the sperm receptor
BUT THAT’S NOT WHAT HAPPENED…..

The virus was unleashed
No immune control on it now, so it became lethal and wiped out the entire colony
Many parties don’t want these sorts of results published
BIOTERRORISM
maybe something like this could be constructed for humans?

even though gene cloning is commonplace now, still need to be vigilant regarding potentially disastrous outcomes! e.g., many institutions do not allow single large sections of virus
DNAs to be cloned - worry is that if enough of it is cloned, you could essentially introduce the pathogenic elements to the benign host organism, thereby making it pathogenic. Worse yet, a new (more pathogenic?) variant of the virus may be inadvertently produced this way. Cloning systems:
Eukaryotes vs Prokaryotes - Chapter 2




fundamental differences in the way DNA is used has implications for production of the desired product “Prokaryotes”

Eukaryotes

usually single chromosome (haploid)

typically diploid or polyploid

usually circular chromosome

linear chromosome

plasmids carried by many

no plasmids (except some yeast)

no nucleus

nucleus

transcription/translation coupled

transcription/translation uncoupled

polycistronic mRNA common (operons)

monocistronic (some exceptions)

1ary transcript = mRNA (no introns)

1ary transcript processed (introns)

mRNA unmodified

5’ methylated cap and 3’poly A tail

initiator tRNA carries N-f-methionine

initiator tRNA carries methionine

initiation of translation at RBS

ribosome binds at 5’cap, scans mRNA to
AUG (and Kosak sequence in mammals), then begins

restriction/modification system present

absent

1 origin of DNA replication

many

Prokaryotic gene structure Fig 2.10 pg 23

Eukaryotic gene structure Fig 2.11 pg 24

Not all eukaryotic genes have introns!
- don’t make the mistake of assuming this

what’s a gene?

what’s a gene?

(this will get messy)

what’s a gene?
P

what’s a gene?
P

OH

what’s a gene?
5’ P

OH 3’

what’s a gene?
5’ P
3’OH

OH 3’
P 5’

what’s a gene?
5’ P
3’OH

OH 3’
P 5’ transcription what’s a gene?
5’ P
3’OH

promoter

OH 3’
P 5’

P transcription what’s a gene?
5’ P
3’OH

promoter
P

transcriptional terminator
OH 3’
T
P 5’ transcription what’s a gene?
5’ P
3’OH

promoter
P

transcriptional terminator
OH 3’
T
P 5’

gene
A discrete DNA segment containing the elements needed to produce an RNA or protein molecule

majority produce a protein (structural genes)

small number produce RNA end­products

e.g. ribosomal RNA (rRNA), transfer RNA (tRNA), siRNA species
…..and other RNAs that have as yet unknown functions

what’s a gene?
5’ P
3’OH

P

T transcription Where does the transcript actually begin?

OH 3’
P 5’

what’s a gene?
5’ P
3’OH

P

T transcription OH 3’
P 5’

what’s a gene?
5’ P
3’OH

P
+1 transcription

T transcription RNA

OH 3’
P 5’

what’s a gene?
5’ P
3’OH

P
+1 transcription

T

OH 3’
P 5’

transcription primary transcript or mRNA RNA

what’s a gene?
5’ P
3’OH

P
+1 transcription

T transcription mRNA

E. coli

OH 3’
P 5’

what’s a gene?
5’ P
3’OH

P
+1 transcription

T

OH 3’
P 5’

transcription mRNA

E. coli

Coding strand? ­ ask yourself which direction the

polymerase is traveling in, and which strand it is using as template

what’s a gene?
5’ P
3’OH

P

T transcription mRNA

OH 3’
P 5’

what’s a gene?
5’ P
3’OH

P

T transcription mRNA

What’s the orientation of the mRNA?

OH 3’
P 5’

+ strand (sense, coding strand)

what’s a gene?
5’ P
3’OH

­ strand (nonsense, non­coding,

OH 3’
P 5’

template)

P

T transcription mRNA
5’ P

3’OH

+ strand (sense, coding strand)

what’s a gene?
5’ P
3’OH

­ strand (nonsense, non­coding,

OH 3’
P 5’

template)

P

T transcription mRNA
5’ P

3’OH translation + strand (sense, coding strand)

what’s a gene?
5’ P
3’OH

­ strand (nonsense, non­coding,

OH 3’
P 5’

template)

P

T transcription ribosome binding site
(E. coli =Shine­Dalgarno to
AUG)
5’ P SD

mRNA
3’OH
translation

+ strand

what’s a gene?
5’ P
3’OH

­ strand SD

P

OH 3’
P 5’

T transcription mRNA

5’ P

3’OH

SD

translation

+ strand

what’s a gene?
5’ P
3’OH

­ strand SD

P

OH 3’
P 5’

T transcription 5’ P

SD

­ start

~6­10 bases apart

translation

3’OH

+ strand

what’s a gene?
5’ P
3’OH

­ strand SD

P

OH 3’
P 5’

T transcription 5’ P

SD

AUG ­ start

translation
What binds this?

3’OH

+ strand

what’s a gene?
5’ P
3’OH

­ strand SD

P

OH 3’
P 5’

T transcription 5’ P
30S
subunit

SD

AUG ­ start

RBS

translation

3’OH

+ strand

what’s a gene?
5’ P
3’OH

­ strand SD

P

OH 3’
P 5’

T transcription 5’ P

SD

AUG ­ start

RBS

translation

3’OH

+ strand

what’s a gene?
5’ P
3’OH

­ strand SD

P

OH 3’
P 5’

T transcription f­Methionine
5’ P

AUG

3’OH

SD

translation

+ strand

what’s a gene?
5’ P
3’OH

­ strand SD

P

ATG

OH 3’
P 5’

T transcription fM
5’ P

AUG

3’OH

SD

translation
What stops translation?

+ strand

what’s a gene?
5’ P
3’OH

­ strand SD

P

ATG

OH 3’
P 5’

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD

translation

nonsense (stop)

3’OH

what’s a gene?
5’ P
3’OH

SD

P

ATG

TAG
TAA
TGA

OH 3’
P 5’

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD

translation

nonsense (stop)

3’OH

what’s a gene?
5’ P
3’OH

SD

P

ATG

TAG
TAA
TGA

OH 3’
P 5’

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD

translation

nonsense (stop)

3’OH

what’s a gene?
5’ P
3’OH

SD

P

ATG

TAG
TAA
TGA

OH 3’
P 5’

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD
5’ UTR

translation

nonsense (stop)

3’OH

what’s a gene?
5’ P
3’OH

SD

P

ATG

TAG
TAA
TGA

OH 3’
P 5’

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD
5’ UTR

translation

nonsense (stop)

3’OH

what’s a gene?
5’ P
3’OH

SD

P

ATG

TAG
TAA
TGA

OH 3’
P 5’

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD
5’ UTR

translation 3’ UTR

nonsense (stop)

3’OH

what’s a gene?
5’ P
3’OH

SD

P

ATG

TAG
TAA
TGA

OH 3’
P 5’

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD
5’ UTR

nonsense (stop)

3’OH

translation 3’ UTR

Monocistronic ­ only 1 gene is represented in the transcript

what’s a gene?
5’ P
3’OH

SD

P

ATG

TAG
TAA
TGA

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD
5’ UTR

OH 3’
P 5’

translation 3’ UTR

Open Reading Frame
(ORF) or Coding
Sequence (CDS)
= protein coding region nonsense (stop)

3’OH

what’s a gene?
5’ P
3’OH

SD

P

ATG

TAG
TAA
TGA

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD
5’ UTR

OH 3’
P 5’

translation 3’ UTR

protein

Open Reading Frame
(ORF) or Coding
Sequence (CDS)
= protein coding region nonsense (stop)

3’OH

what’s a gene?
5’ P
3’OH

SD

P

ATG

TAG
TAA
TGA

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD
5’ UTR

OH 3’
P 5’

translation 3’ UTR

NH2 protein Open Reading Frame
(ORF) or Coding
Sequence (CDS)
= protein coding region nonsense (stop)

3’OH

what’s a gene?
5’ P
3’OH

ATG

SD

P

TAG
TAA
TGA

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD
5’ UTR

OH 3’
P 5’

translation 3’ UTR

NH2fM protein Open Reading Frame
(ORF) or Coding
Sequence (CDS)
= protein coding region nonsense (stop)

3’OH

what’s a gene?
5’ P
3’OH

ATG

SD

P

TAG
TAA
TGA

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD
5’ UTR

translation 3’ UTR

NH2fM
COOH

OH 3’
P 5’

protein

Open Reading Frame
(ORF) or Coding
Sequence (CDS)
= protein coding region nonsense (stop)

3’OH

what’s a gene?
5’ P
3’OH

ATG

SD

P

TAG
TAA
TGA

T transcription fM
5’ P

AUG

UAG
UAA
UGA

SD
5’ UTR

translation 3’ UTR

NH2fM
COOH

OH 3’
P 5’

protein

Open Reading Frame
(ORF) or Coding
Sequence (CDS)
= protein coding region nonsense (stop)

3’OH

Bacterial mRNA
What’s wrong?
5’
P

3’
RBS

SD

ATG

UAG

T

Tutorial 1

If I give you the protein, can you walk the steps backwards to show me the important elements in the transcript and in the original DNA strand?

Tutorial topics are posted on LEARN so you can see what you’re in for!

mRNA processing in Eukaryotes posttranscriptional modifications of mRNA
5’guanine cap

loading signal for ribosome

necessary for translation

cannot normally translate polycistronic mRNA
3’polyadenine tail

contributes to stability

needed for translation
Fig 2.11 pg 24

mRNA processing in Eukaryotes posttranscriptional modifications of mRNA introns/exons ● primary transcript undergoes splicing events to remove introns and ligate exons together

Prokaryotic gene arrangements
- polycistronic mRNA and operons

Prokaryotic gene arrangements: is this an operon? polycistronic RNA = mRNA that encodes two or more proteins

DNA

P

mRNA

proteins

ATG

TAG

SD AUG

UAG

ATG

TAA

AUG

UAA

SD protein 1 ORF protein 2 ORF
SD

SD

T

T

Prokaryotic gene arrangements operon = a cluster of genes that are coordinately regulated and under the control of an operator.
DNA

P

O

ATG

TAG

ATG

TAA

SD protein 1 ORF protein 2 ORF
SD

T

…. Here’s where the definition of gene becomes tricky
Are there 2 genes shown here or just one gene that encodes
2 proteins?

Prokaryotic gene arrangements operon = a cluster of genes that are coordinately regulated and under the control of an operator.
DNA

P

O

ATG

TAG

ATG

TAA

SD protein 1 ORF protein 2 ORF
SD

repressor protein no transcription

T

Prokaryotic gene arrangements operon = a cluster of genes that are coordinately regulated and under the control of an operator.
DNA

P

O

ATG

TAG

ATG

TAA

SD protein 1 ORF protein 2 ORF
SD

T

repressor protein no transcription

BUT!!
In the presence of the inducing substance...

Prokaryotic gene arrangements

DNA

P

O

ATG

TAG

SD AUG

UAG

ATG

TAA

AUG

UAA T

SD protein 1 ORF protein 2 ORF
SD

T

inducer repressor protein

polycistronic mRNA proteins

SD

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