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A quick look at mRNA Splice Variants

-Beadle and Tatum Redux

-Beadle and Tatum Redux

In my microbiology class this past week, we were discussing how prokaryotes and eukaryotes differ in their handling of DNA, RNA and gene regulation. Mostly, we focused on how the presence of the nuclear membrane in eukaryotes separates the processes of transcription and translation and what this results in. Briefly, bacteria are prokaryotic life forms that lack a defined nucleus (among other differences). Because of this, when bacteria transcribe mRNA, it is immediately available for translation – the DNA, RNA polymerase and Ribosome all exist in shared space. Below is a classic image of a strand of DNA(stretching left to right) in E. coli being transcribed into RNA. The RNA molecules extend away from the DNA and appear to travel up or down away from the DNA in this micrograph. Along the length of the RNA, we see dense ribosomes which are busy synthesizing proteins.

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In Eukaryotes, the nucleus encases the DNA, the RNA polymerases and mRNA. mRNA can be completely synthesized and modified in a number of ways before they are exported from the nucleus to the cytoplasm, where ribosomes will translate the message into protein.

One of the modifications of Eukaryotic mRNA we spoke about was splicing. Splicing is a means of snipping segments of non-coding introns out of the mRNA leaving a mature mRNA with a continuous strand of exons. One interesting possibility this enables is the production of alternative sequences made from differential splicing of the immature mRNA. These alternative mRNAs are known as splice variants. At this point, I was asked for an example of a gene that is handled in this way and was caught flat-footed.

Hmm. Perhaps this is something that I’d heard so much about in classes but never in the ‘real world’. I’ll have to look.

One of the first things I found was this discussion of splice variants suggesting that this was not a biologically significant event. i.e., the RNA may be alternatively spliced, but do these splice variants actually result in functional proteins with different properties. The author poses a challenge to find examples of splice variants that are ‘real’. The ensuing discussion is a good one.

What would this looks like?

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Regardless, I found a paper with some good figures that may help students understand how this phenomenon (at least putatively) occurs.  Here’s the best figure presenting a diagram of the different mRNAs created and gels and sequence data indicating that these exist.

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The above Figure shows the presence of distinct RNA species, although that, alone, does not mean that these RNA are ever made into protein. To do that, western blots of protein extracted from various tissues is shown below.

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What is left to find is whether each of these two proteins actually does something. Are both forms required? Are their functions distinguishable?
My quick look through the literature did not uncover any evidence for this last question. If anyone out there knows the literature on this, I would love a push in the right direction. It doesn’t matter what gene we’re looking at, just that it is an example of alternative splicing and that each of the splice variants is actually made and has some identifiable and distinct function.
 
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Posted by on April 4, 2014 in Uncategorized

 

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reviewing TEAS test questions

In preparing for a new semester, I’ve been downloading a few free question compilations for the TEAS test. TEAS is the ‘Test of Essential Academic Skills’ that students wishing to enter into nursing programs must take. Since many of my students in both my Biology and Microbiology classes wish to enter nursing programs and are taking required classes for that purpose, I find these questions to be very useful in directing my syllabus creation.

However, today, I found this question amongst the free samples offered at the practicequiz.com website:

Category – Science

Q – What is a term used to describe the subtle energy running through the body?
A. The Tao
B. The Force
C. Chi
D. ATP
Your answer: –
Answer: C – Chi is a term used for the subtle energy that runs through the body.
Chi is specific to Traditional Chinese Medicine. Other terms used are ki, prana and life force.

What am I to think?

I’m not sure that I am comfortable with ‘Chi’ being a legitimate concept being taught in science classes.

Am I wrong to think that the science and medicine taught to students of these disciplines should be objectively verifiable? Is ‘Chi’ a documented phenomenon? Or is this just a word / description that describes some metaphysical concept?

Wikipedia uses the following to define Chi:

Qi is the central underlying principle in traditional Chinese medicine and martial arts…Elements of the qi concept can also be found in Western popular culture, for example “The Force” in Star Wars.”

Given this loose definition for an equally loose concept, I think “The Force” is an equally reasonable answer to the question as it is posed. And no answer to this question should be acceptable or expected in a serious exam. I certainly hope that this question is an example made up by practicequiz.com, and not one that my students are ever likely to see on a real examination.

See this link for a review of chi, also known as qi or qidong, in medicine.

 
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Posted by on December 26, 2013 in Uncategorized

 

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Vitamin Supplements: Vital? or Rubbish?

ImageIn class today, I was asked what I knew about vitamin supplements and whether it was a good idea to take them or not. To follow up on that conversation, I thought I would post a link to an article that addresses this question directly. This article, by Paul Offit, was published by the Atlantic magazine July 19 of this year and deals with some of the same material from his newest book, Do You Believe in Magic?

Vitamines are certainly required for healthy living, something reflected by their very name (vitamin = vital amine). The question is, how much of each of these do we need to add above and and beyond what we get from a well-balanced diet? It has been proposed that excessive doses of certain vitamins could be a panacea leading to elimination of many of life’s ailments.  From the Atlantic article…

In 1970, [Nobel Prize winning scientist, Linus] Pauling published Vitamin C and the Common Cold, urging the public to take 3,000 milligrams of vitamin C every day (about 50 times the recommended daily allowance). Pauling believed that the common cold would soon be a historical footnote. “It will take decades to eradicate the common cold completely,” he wrote, “but it can, I believe, be controlled entirely in the United States and some other countries within a few years. I look forward to witnessing this step toward a better world.” Pauling’s book became an instant best seller.

ImageThese pronouncements would have amounted to nothing coming from many other people, but Pauling was not just any other person. There’s a chance his pair of Nobel Prizes may have held some weight in people’s minds. 

 
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Posted by on November 14, 2013 in Uncategorized

 

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Because it was on Dr. Oz, I’m more likely to think it’s a scam

doctor-ozI got something interesting in my inbox the other day. Something that I assume was a  friend’s email address getting hacked – although it’s the least offensive (apparent) hack I’ve ever seen (he says as the viruses circulate around his computer’s RAM).

It was a nearly blank email with a link to a Dr. Oz clip about the weight-loss promoting effects of green coffee extract, which contains high concentrations of chlorogenic acids. These molecules are said to promote weight loss through increasing metabolism.

Being a scientist means being a skeptic. In this case, because I already feel like it must be BS due to its connection with Dr. Oz (an Oprah-elevated proponent of many untested, ‘alternative’ therapies), the challenge for me is to admit the possibility that this stuff may work. So, rather than looking through the data to see if there’s anything to deny the claim, I’m really trying hard to look at the data to see any glimmer  of possibility.

Here’s a link to the Dr. Oz article online. The article was published in the January 2012  Diabetes, Metabolic Syndrome and Obesity, and happily the entire article is available free of charge. So let’s look at the data…

The article examines a “22-week crossover study was conducted to examine the efficacy and safety of a commercial green coffee extract product GCA™ at reducing weight and body mass in 16 overweight adults.” Half of the participants were male and half female – a typical study setup (although I do worry about how data is handled when looking at both sexes together, so let’s pay attention to that.)

Dr. Oz’s website indicates that “The subjects (taking the supplement) lost an average of almost 18 pounds – this was 10% of their overall body weight and 4.4% of their overall body fat.” These are pretty hefty claims, but I could use losing 18lbs, so let’s see where this goes.

The study followed those eight men and eight women for 22 weeks. At the beginning of the study, the average body mass index (BMI) at the start of the study was 28.22 ±  0.91 kg/m2 . Determine your own BMI here.

Note that BMI < 18.5 is underweight

18.5  –  25     healthy weight

25   –   30      overweight

30+               obese

This puts the study participants at the high end of overweight, but ‘preobese’.

Dosages of the green coffee extract and placebo were as follows:

“This study utilized two dosage levels of GCA, as well as a placebo. The high-dose condition was 350 mg of GCA taken orally three times daily. The low-dose condition was 350 mg of GCA taken orally twice daily. The placebo condition consisted of a 350 mg inert capsule of an inactive substance taken orally three times daily.”

I don’t think I’m the first one to point out that it’s hard to have a double blind trial when the dosages are distinguishable (two times vs three times daily). At least the placebo should be indistinguishable from the high dose.

One early eye-catching piece of data is from Table I, that summarizes the data of all precipitants as

BMI (kg/m2) pre study:28.22 ± 0.91        post study:25.25 ± 1.19     change-2.92 ± 0.85**, -10.3%

On average, all subjects lost weight during the study. But this really tells us nothing because we could see a 10% drop in BMI if the test arm lost 20% and then placebo arm stayed the same, or we could see the same thing if the weight loss occurred during ALL arms of the study.

Perhaps this reporting of data is justified by the next statement that participants all rotated through being on high dose, lose dose or placebo with intervening washout periods. Presumably, this makes the most of a small sampling of people, but I do find it harder to be confident about the data. Then again, I have never been involved in any human trial of this kind.

here’s the data:

High Dose arm:

start    BMI (kg/m2) 26.78 ± 1.55  –>    end 26.03 ± 1.36

Low Dose arm:

start    BMI (kg/m2) 26.25 ± 1.37  –>    end 25.66 ± 1.20

placebo arm:

start    BMI (kg/m2) 25.66 ± 1.20  –>  and 26.67 ± 1.72

At first glance this might appear to be pretty good. But let’s graph it out:

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the data continue to look great.

Now, with error bars:

ImageHuh. Not so hot anymore.

Also, I’m not how sure this was done, but they get p values for HD p = 0.002, LD p = 0.003, placebo p = 0.384. These stats mean that the HD and LD groups are showing very significant differences, while the placebo group is not. You should be able to see this in the graph with error bars (as an approximation of significance). Unfortunately, I see a whole lot of no nothing. But, perhaps BMI is not the appropriate way to observe weight change (we are, after all not seeing specific weight changes, but changes within a group, i.e. diversity)

Another way to try to see what’s going on is to take a look at the weight data:

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The data were presented in a number of other ways, but each of these was confusing and didn’t illustrate any clear conclusion (my interpretation).If the individuals’ data were visualized as a scatter plot, this might show us something – or data for each individuals change while in each group… As it is, we see unclear data with spectacular statistics, but we don’t get to see enough to be convinced of the changes.

Rather than go on and get more and more skeptical, let’s say, although we don’t see a lot here, the data,as reported, would make us want to see a larger study with some revisions for control of diet, exercise monitoring and a change in the way osage is administered so as to maintain the ‘blindness’ of the study.

 
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Posted by on July 22, 2013 in Uncategorized

 

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