Lecture - Introduction to the course

Today we continued with the introduction we started on Monday.  We talked about some generic information about the structure and function of nucleic acids, the processes of transcription and translation (make sure to know the difference!), mechanisms of control of gene expression, and talked a little about the reaches and developments of recombinant DNA technology.  We even mentioned what the field of genomics and proteomics can do.

Next class: On Monday we will be talking about chromosomes (p. 233-244) and hopefully also about genome evolution (p. 245-260).

----------------------

Lab 02 - Restriction Enzyme Digestions (REDs) and analysis of lambda DNA

A micrograph of multiple bacteriopages

Restriction enzymes are one of the most basic and important tools in molecular biology. They evolved in bacteria to attack and cut (cleave) foreign DNA, mostly from bacteriophages (viruses that "eat" bacteria). But hey have been isolated to be used in the lab, and are useful to cut any kind of DNA, not just viral.

Cleaving DNA is the first step in any technique that involves recombinant DNA technology. There are techniques that use special enzymes to paste (ligate) different fragments of DNA. For instance a gene can be ligated into a plasmid that can be inserted into bacteria to make many copies of it via bacterial reproduction (cloning), something we will do in a few weeks.

Today we used lambda DNA (DNA from the common lambda bacteriophage) as the substrate to be cleaved with three different restriction enzymes: EcoRI, HindIII, and PstI.

We then ran our first agarose gel electrophoreses of the quarter (there shall be many more), and here are the results. Click on each pic to get a full size image of each gel:

For the lab report:

• The lab report is due Thursday Dec 11th.
• If you can answer the questions on page 44 of "exercise 3" in the lab manual you'll have a solid discussion of results section. Use them as a guide to get ideas to discuss.
• In one of the four posted pictures the samples are significantly smeared. In your discussion include your thoughts (or findings) on what may have caused this.
  
• Include a picture of your gel. If you turn in the one printed in the lab (on thermal paper) you can label the lanes with an ultrafine point permanent marker (sharpies and the like).
• A pdf file of a semi-log paper page will be e-mailed to you for your convinience.
  

Research questions

• The gels we used were pre-poured for us. They were 1% agarose gels. What does this mean?
• If you want to improve the resolution of the gel (making bands sharper, improving separation of bands of similar size) what can you do different when preparing the gel?

----------------------

Lab 01 - Playing with micropipettes

Today we had our first lab meeting of the quarter.  Lab manuals, lab notebooks, "blue books" (for lecture quizzes), and disposable lab coats were distributed.   We talked about how to behave in the lab and went over the first and most simple lab exercise:  getting acquainted (or re-acquainted) with micropipettes.  The report for this lab was handed in at the very end of the lab.

Tomorrow:  Lab 2 ("Exercise 3" in the lab manual), Restriction Enzyme Digests (REDs) and gel electrophoreses.

------------------------------

Winter 08-09 quarter

Welcome to the Winter 08-09 Intro to Molecular Biology class at ONU...!

This blog is meant to be a tool to improve communication between students and the instructor of the class (Alonso Córdoba) and to keep track of the actual happenings in the class throughout the quarter.  Feel free to browse through old entries (Fall 08 quarter) to get an idea of the kind of information you can get by reading this blog.  

An interesting feature is the fact that you can find questions and answers to old quizzes.  You can use this tool to get used to the quiz format or even to study for upcoming quizzes and exams.

Is there something you would like to see posted on this blog?  Just let me know.  I am open to new ideas, suggestions, and constructive criticism.  Just drop me a line or give me a call (contact info on the column to the right of entries).

I hope you enjoy this quarter...!  (stay warm!)

-----------------------

Random pics... (a Fall 2008 lab)

A little Ice Man? I clearly didn't give students enough to do that day in lab... I'll have to be a little tougher in the future....

------------------------

Final exam

The final exam took place between 8:00 and 10:00 am at Meyer Hall 128.

It is worth 100 points. There were 80 multiple-choice questions, worth 1 point each, and 6 short essay questions. 4 questions were worth 4 points, and 2 of them were worth 2 points.

Exam stats

• Mean score: 79 (exam 1: 86; exam 2: 79)
  
• Standard deviation: 15.3 (exam 1: 8.8; exam 2: 10.9)
  
• Mode: 77 (exam 1: 90; exam 2: 82)
  

Although the average was steady compared to exam 2, the mode decreased and the standard deviation increased. The likely reason is that the new material that was heavily weighted compared with exam 1 was difficult for some people. But some people had very high scores. Maybe studying more at the beginning of the semester crated a solid base to build upon?

Score frequency distribution

Toughest questions (statistically):

13 & 20. If two genes in an individual are derived from a single ancestral gene, they are called
c & d. Paralogs

Comment: The questions were phrased slightly differently, but the essence of the question and the answer were the same. If an indidual has two (or more) genes in its genome, originated from gene duplications, the are called paralogs or paralogous genes. Orthologs or orthologous genes, the alternative, are genes, in genomes of different species, that have evolved from the same gene in an ancestor. They are the same gene, in different species, not a product of a gene duplication.

43. What is an operon?
a. a series of genes transcribed into a single mRNA in prokaryotes

Comment: Not just they are transcribed into a single mRNA, but are controlled by a single promoter. They are not found in eukaryotes, where each individual gene has its own promoter, and it's transcribed into a separate mRNA.


49. What is an mediator?
d. a complex of proteins that function as an intermediary between transcription factors and RNA polymerase

Comment: There are cases, in complex genetic switches, when having the right transcription factors in place is not enough to start trasncription. The mediator (a complex of 24 subunits), acts as an intermediary (or a coordinator, if you will) between them and the polymerase. It's not a transcription factor per se, but just as important.


57. What is an gene control region?
b. a region of DNA containing elements that control and initiate gene transcription

Comment: The DNA control region can be even longer than the actal gene (introns included). It has several elements that will have to act in coordination to start gene transcription. The gene control region is what makes complex genetic switches complex.


72. If mutation rate cannot be masured experimentally it can be estimated by...
c. comparing the genomes of multiple species

Comment: When generation times are too long to wait around and see how high mutation rate is, the only option is estimating it from the differences found in sequence alignments. There are algorithms that allow the use of nucleotide or amino acid sequence differences as the basis dor such calculation.


84. Describe RNAi. Include the steps in the process and explain its goal
Read pages 493-496 in the text book

Comment: Even though we didn't cover it in class, we did talk about the importance of such process for the cell and in terms of a research tool. I did tell you to read about it and it was specifically mentioned in the study guide as one of the potential topics for open questions.
RNA interference (RNAi) has become an imnportant molecular technique to study patterns of gene expression.

---------------------

Course evaluations

Here's the website for filling in the course evaluations:

http://onuapp1.onu.edu/courseval/ets/et.asp?nxappid=WCQ&nxmid=start

Use your user name and the last 4 digits of your social as your password.  At least that's what it's supposed to be.  If it's not, they will e-mail you the correct password.

Update (Nov 17 08):  22 out of 24 people filled in course evaluations

------------------------------ 

Meeting @ McIntosh - Last presentations + class conversation

We met at McIntosh 204 instead of at the regular lab to be able to use a projector and have the last 4 talks of Tuesday's symposium:

_____________________________________________________

TRANSGENIC PLANT VACCINES: A REVIEW FROM BOTANICAL AND HUMAN PERSPECTIVES
Josh Holloway and Eric Schultz

MODERN ADVANCEMENTS IN THE PRODUCTION AND SCREENING OF GENETICALLY MODIFIED CROPS
Stuart Collins and Matt Vemich

THE USE OF DNA IN RELEASING INNOCENT PRISONERS
Brandon Vieira and John Jacobs

RECOMBINANT DNA TECHNOLOGIES IN FORENSIC SCIENCES
Katie Elsass and Jonathan Digby
_____________________________________________________

After the talks, each lab section had a separate meeting to talk about impressions about the class and how it could be improved to accommodate the needs of students. Students gave input and made several recommendations, most of which will be implemented in the winter quarter.

-------------------------

MEETING AT McINTOSH 204 - Thursday the 13th

Tomorrow, Thursday the 13th, we will be meeting at the McINTOSH CENTER, CONFERENCE ROOM 204!

We will meet at the regular lab times to finish the remaining presentations and have a little bit of lecture.

Presentations section 1 (8:00 am):

• Josh Holloway and Eric Schultz
• Stuart Collins and Matt Vemich

Presentations section 2 (10:00 am):

• Brandon Vieira and John Jacobs
• Jonathan Digby and Katie Elsass

-------------------------

Introductory Molecular Biology Symposium

This symposium featured students in the class presenting the results of their bibliographic research summarized in review papers on molecular biology topics. Papers will be compiled in the proceedings for the symposium.

Presentations, ordered as scheduled, were:

__________________________________________________

PLANT DEFENSE MECHANISMS : AN UNEXPECTED ARSENAL
Josh Judkins and Jackie Champa

ETIOLOGY AND EVOLUTION OF MRSA
Katie Somes and CJ Jewel

THE USE OF DNA IN FORENSICS
Mike Breckenridge and David Cassidy

GENETIC MODIFICATION IN HUMANS
Amy Mattern and Ryan Wheaton

DNA FINGERPRINTING OF DISASTER VICTIMS
Anessa Storer and Chloé Seiller

GENE THERAPEUTICS: A BRIEF REVIEW OF THE HISTORY, TECHNIQUES, AND APPLICATIONS OF A POTENTIALLY DISEASE-CURING PROCESS
Kyle Stinehart and Courtney Zupancic

TRANSGENIC PLANT VACCINES: A REVIEW FROM BOTANICAL AND HUMAN PERSPECTIVES
Josh Holloway and Eric Schultz

CUSTOM DRUGS BASED ON GENETIC PROFILE
Amanda Blandford and Jackie Trumpower

MODERN ADVANCEMENTS IN THE PRODUCTION AND SCREENING OF GENETICALLY MODIFIED CROPS
Stuart Collins and Matt Vemich

EFFECTS AND DETECTION OF DIGEORGE SYNDROME (22Q11.2 DELETION SYNDROME)
Phil Schulze and Krystal DeMonte

THE USE OF DNA IN RELEASING INNOCENT PRISONERS
Brandon Vieira and John Jacobs

RECOMBINANT DNA TECHNOLOGIES IN FORENSIC SCIENCES
Katie Elsass and Jonathan Digby
___________________________________________________

Complex genetic switches and post-transcriptional control

We covered pages 439-447 regarding complex genetic switches, specifically in Eukaryota. We did a short comparison between prokaryot and eukaryot genetic switches. The concept of mediator, absent in prokaryots but present in eukaryots was introduced.

We studied a typical eukaryotic gene control region. Several elements within such region were discussed (spacers, regulatory sequences, TATA box, promoter), as well as related proteins (regulatory proteins, general trasncription factors, RNA polymerase II).

On pages 477-497 post-transcriptional gene expression controls were discussed. Our discussion was not detailed but we talked about transcription attenuation, riboswitches, alternative RNA splicing, RNA editing, and RNA interference (RNAi).



Quiz #14 Q&As

1. In DNA-Binding proteins, what is the difference between α-helices and β-sheets? (other than the helical and non-helical structure)
An α-helix is made of a single polypeptide. β-sheets are made by several polypeptides

2. What is a promoter?
A region of regualtory DNA where RNA polymerase binds

3. What is an operon?
A series of adjacent genes controlled by a single promoter

4. What is an operator?
A short regulatory DNA sequence within a promoter recognized by a represor

5. What is a complex genetic “switch”?
A a genetic switch controlled by multiple gene regulatory poteins

6. What is a repressor?
A protein that binds to an operator to repress the expression of a gene

7. What is an activator? (hint: it does more than just turning a gene on)
It's a protein that increases the transcription efficiency of a gene

----------------------------

LAB - Bioinformatics

DNA sequence alignment viewed with the multiple alignment editor Se-Al. On the far right there are insertions and/or deletions in several sequences (click pic for full-size image)

We had laptops for each student in the lab and had a small and crude (quick and dirty?) overview of some of the few tools available in bioinformatics.

The main focus of our exercise was the use of the algorithm known as BLAST (Basic Local Alignment Search Tool), available at the NCBI webpage. After an overview of some of the main features of the site (which is updated VERY frequently), we "BLASTed" (used BLAST) a few unknown protein (using 'protein BLAST') and nucleotide (using 'nucleotide BLAST') sequences. We found out what the sequences were and to what organisms they belonged. This is one of many possible uses of the BLAST algorithm.

During this lab students were also introduced to the fasta file format and its use for sequence alignment. After finding out what our problem nucleic acid sequences were (by BLASTing)and to what organisms they belonged we made a fasta file with all of them and aligned them using an on-line version of ClustalW available on the European Bioinformatics Institute website.

The following learning outcomes should have been met:

• Introduction to the concept and field of bioinformatics
• Introduction to the main sequence data repository in the Americas and one of the main in the world: NCBI
• Introduction to the main database in the NCBI website: GenBank
• Basic understanding of the fasta file format
• Basic use of the BLAST algorithm
• Introduction to the concept of sequence alignment

Lab report

The lab report can be typed and it won't follow the format of the previous reports. Please include:

• An brief introduction explaining what bioinformatics is
• A summary of the exercises performed in the lab (methods)
  
• Findings about the problem sequences. What organisms did they belong to? What proteins and genes were they? - Include the three first hits for each search if available. No need to include the actual sequences
• Research question: What are the potential uses of a sequence alignment?

---------------------------

LAB - Silver staining a polyacrylamide gel

Download the Bio-Rad Silver Stain Plus protocol in pdf format by clicking here.

pGLO plasmid restriction digest, PAGE, and visualization through silver staining.
The staining procedure didn't work as expected (due to longer than needed staining and shorter than required drying...), so no picture can be taken. Here are drawings of the band patterns:

• How many bands did you expect based on the number of restriction sites for BamH I? (check your plasmid map)
• What may explain the band pattern you got?

Speculate about these questions in your discussion.

------------------------------------

Exam 02

In the exam 80% of the questions were multiple choice, with the remaining 20% being short essay questions. I decided to include essay questions becuase people had not been doing very well in quizzes, but this section was actually the strongest for most people.

Exam stats:

• Mean score: 79 (86 in exam 1)
• Standard deviation: 10.9 (8.8 in exam 1)
• Mode: 82 (90 in exam 1)

Scores were down in average and the standard deviation increased compared to exam 1. Most likely because of the material being newer for most students, and a little more complex. Any other hypothesis?

Score frequency distribution

Toughest questions (statistically):

12. Mutation rate can be measured experimentally...
b. comparing the genomes of an ancestor and its descendants

Comment: A bacterial genome can be sequenced, then culturing the strain for a number of generations, and then sequencing the genome of a descendant. By comparing both genome sequences and knowing the number of generations past, the mutation rate can be measured.


13. Mutation rate can be masured by observation...
c. comparing the genomes of multiple species

Comment: When generation times are too long to actually allow many generations to go by (e.g. in mammals) an experimental measurement of mutation rate is not possible. In such cases it can be estimated by comparing the genomes of multiple related species and measuring generic distances.


20. A primer strand...
b. is the strand of DNA being extended

Comment: Primer strand is the technical term for a "daughter" strand, whereas template strand is the name for the "parent" strand. Most people circled the answer for 'what is a primer'. A primer is a strand of oligonucleotides, but it's not called 'primer strand'. Just 'primer'.


25. Single strand DNA-binding proteins...
e. straighten the template strand during relpication

Comment: They prevent the single tremplate strand of forming loops that can interfere with the reading of the DNA plymerase.

-----------------------------

DNA-Binding proteins and genetic switches

We covered pages 416-432 of the book, dealing with the properties that gene regulatory proteins have, mainly the fact that they can read the information in the DNA double helix without mamking direct contact with the bases, but by 'reading' chemical signals in the phosphodiester backbone unique to each nucleotide.

We also talked about the main structural motifs that are common to gene regulatory proteins (helix-turn-helix, zinc finger beta-sheet, leucine zipper, and helix-loop-helix).

We also covered pages 432-439, explaining how simple genetic switches work. They have been studied in bacteria and have the particularity that are composed by a single gene regulatory protein. We introduced the concepts of operon, operator, activator, and repressor.

Next class: Complex genetic switches (in eukaryotes), and post-trancriptional control

Quiz #13 Q&As:

1. In a multicellular organism what process ensures that the only proteins and RNAs to be produced in a particular cell are those that that particular cell (and, therefore, tissues and organs) requires?
Control of gene expression

2. In a multicellular organism most cells have all the information required to build a whole organism. Why does a cell “know” that only a fraction of the information must be used, and not all of it?
Control of gene expression

3. What is transcriptional control?
A mechanism through which gene regulatory proteins activate or repress trasncription of a gene

4. Mention two kinds of control of gene expression (other than transcriptional)
RNA processing, RNA transport and control, translational mRNA degradation, RNA editing, RNA differential splicing...

5. What is the difference between a simple genetic “switch” and a complex one?
In a simple one only one protein turns de gene on or off. In a complex one many proteins are involved, usually hundreds.

-------------------------------

LAB - pGLO Restriction Digest

pGLO plasmid map
click on picture for a full size image

Following the plasmid isolation procedure using minipreps, our next step would be the one we would follow if we were cloning a gene: Isolate the gene from the plasmid. In this case we want to isolate the GFP gene, and we will be using the BamH I restriction enzyme, which cuts GFP from the plasmid, and it also cuts the gene in two fragments.

First we ran an agarose gel to know if we actually have succesfully isolated our plasmid DNA (we do know we have plasmid DNA in some of our section 2 samples, but it is still unclear in our section 1 samples). After that, we set up the restriction enzyme digest (RED).

Next week we will run a polyacrylamyde gel (PAGE) that we will silver stain. Silver staining is a very sensitive technique to visualize proteins and DNA in agarose and polyacrylamide gels.

--------------------------

LAB - Plasmid isolation using mini-preps

We have completed a cycle, and after genetically tranforming bacteria using Bio-Rad's pGLO pasmid, it's time to isolate it from our clones. We used the Wizard® Plus Minipreps DNA Purification System (Promega), which provides a simple and reliable method for rapidly isolating plasmid DNA. This system can be used to isolate any plasmid but works most efficiently when the plasmid is <20,000bp. Click here to view a pdf of the complete protocol of the miniprep kit.

We lysed the bacteria and added a series of reagents to isolate plasmid DNA wothout getting any bacterial genomic DNA. The ultimate objective is to isolate the GFP gene from the rest of the plasmid.

-----------------------------

Control of gene expression

Different levels at which gene control can be exerted
(click on pic for full size image)

We started considering the mechanisms through which cell "knows" what subset of the genetic information must express in order to become the cell that it's supposed to be (a neuron, a lymphocite, a pancreatic cell, etc...). We considered the different levels at which a cell can control what proteins and RNAs are produced.

We also introduced the concept of gene regulatory proteins (a.k.a. transcription factors) and their relation to regulatory DNA sequences., and how they can read the information coded in the DNA without openeing the double helix, based solely on unambiguous chemical properties of the phosphate backbone of each base pair in the major grooves of the helix.

Material covered: pages 411-418

Reading for next class: pages 418-454

Quiz 12 Qs&As:

1. What is DNA repair?
The mechanism to correct changes in DNA sequence after transcription (after proofreading), before the information is passed to daughter cells. It is performed by a battery of enzymes, and not by a single one.

2. Mention or explain (briefly!) a way in which DNA damage can be removed
Base excision repair, nucleotide excision repair

3. What happens to DNA sequence when it is repaired by non-homologous end joining compared to when it is repaired by homologous recombination?
In non-homologous end joining one or more nucleotides of each strand are deleted.
In homologous recombination the information of the missing piece of strand can be copied from the sister chromatid and restored.

4. Which protein detects DNA damage during the transcription process?
RNA polymerase

5. What is control of gene expression?
Biochemical mechanisms to express selected genes and repress those that are not necesary

DNA Repair

Today we had an activity which goal is to improve class participation (and preparation). After doing a quick review of last class, based on the replication "fork" figure (see entry on Wednesday October 22nd - 'DNA replication machinery'), students prepared a 1-2 page essay on today's topic: DNA repair. Students were allowed (and encouraged) to consult the textbook, discuss topics, and ask questions.

The essay will be worth 10 points (to be added to the quiz scores)

Material covered can be found in pages 295-304. Main topics to consider for the exam are:

• Importance of DNA repair
• Repair of the double helix
• Pathways of DNA repair
• DNA polymerases specialized on DNA repair
  
• Repair of double strand breaks
• Effect of DNA repair on the cell cycle

Reading for tomorrow: Control of gene expression (p 411-454... don't freak out. Not every single word will be covered. But read the whole thing if possible)

Quiz #11 Q&As

1. What is a replication fork?
Enzyme induuced 'y' shape conformation of the DNA replicaiton helix

2. What does DNA polymerase do?
It synthesizes mRNAs based on molecualr infromtion

3. What does DNA primase do?
It binds a small RNA promer to the template strand of DNA so the polymerase can start extending the sequence

4. What is DNA repair?
Mechanisms to correct post-proofreading DNA changes, performed by a variety of enzymes

5. How are DNA repair and proofreading different?
Proofreading is done only by some polymerases. DNA repair involves many enzymes

6. What are Okazaki fragments? (bonus: 3 points)
During DNA replication the short fragments of DNA resulting from replication of the lagging strand taking place in the opposite direction to that in which the replication fork opens. They are later stuck together by a DNA ligase to form a continuous strand

-------------------------

Connecting the last few labs

The last several labs have been connected and here's a little summary to remind you how they are interconnected:

• Labs 7-8: Bacterial genetic transformation with pGLO plasmid
• Labs 9-10: Protein (GFP) purification by chromatography
• Labs 11-12: Bradford protein (GFP) determinations
• Lab 13: Native Protein PAGE
• Lab 14: Denaturing Protein PAGE

When we transformed our E. coli with the pGLO plasmid, containing the gene for the Green Fluorescent Protein (GFP) we were also producing the material we were going to use in the next several labs. We transformed the bacteria, made them produce our GFP, then purified that GFP and now we are determining how much of it there is and how big the polypetide is.
This process is analogous to that in the industry to produce a protein for commercial applications asn also abalogous to a research process when a gene for a new protein is being discovered or studied.

Dont think of these few labs as independent ones, but as steps of a single longer lab.

Labs 11-14 can be summarized in a single lab report. Here are the questions for such report:

1. Why do we make our absorbance measurements at 595 nm?
2. When you heat up DNA or protein samples up to 95ºC they get denatured. Specifically what happens to DNA and proteins? In other words what is DNA denaturation and what is protein denaturation?
3. When estimating protein size would you rely more on the native of the denaturing gel results? Why?
4. Some groups had more than one protein (more than just GFP) in their gels. HOw do you explain this?
   

----------------------------