Author Archives: Floyd A. Reed

Polytene Chromosome Imaging

One undergraduate in the lab, Kenton Asao, is interested in chromosome evolution and has been troubleshooting protocols to prepare insect polytene chromosomes for imaging.  I worked with polytene chromosomes many years ago in grad school---but not since---so I am quite rusty and need to brush up on this myself, and this is something we would like to be able to routinely do in the lab.  Long story short, it wasn't working starting out so we sent some photos (to make sure we were dissecting out the right tissues) and asked for some advice from Kevin Cook (D. of Biology, Indiana U.) who kindly sent us his protocol.  We continued troubleshooting and we were getting quite frustrated trying different stains and imaging approaches but nothing seemed to be working (we also asked for advice from Gert de Couet in the department here at U.H. who loaned us some different dye and reagents to try out).  In the meantime we tried extracting DNA and doing some PCR for some damsel flies and that didn't even work...!  Then this morning Kenton found me and said that he needed to show me something.  After taking a look I went back to my office to get our microscope camera and set it up to record some images.

Some background.  Generally chromosomes are very tiny and difficult to image with standard equipment.  However, some cells undergo endoreduplication where the chromosomes divide but the cells do not.  In some of our muscle and liver cells there are actually four (tetraploid) or eight (octoploid) copies of each chromosome instead of the normal two copies (diploid--one from each parent) that we are used to thinking about.  In older fly larvae that are about to pupate and metamorphose into adults the salivary gland chromosomes divide approximately nine times resulting in 512 copies of each of two starting chromosome copies or about 1,024 total copies per cell.  In addition they stay physically associated with each other so that they form large (gigantic on a cellular level) structures.  Each individual chromosome is made up of about half DNA (actually a single very long strand of DNA) and half proteins that organize the DNA into the structure of the chromsome.  The density and composition of proteins changes along the chromosome so that different regions have different staining levels that result in a banding pattern.  In polytene chromosomes all 1,000+ copies are organized together into these large structures.

This first image below is of some cells in larval salivary glands under the microscope.  Within the cells you can make out the nuclei and maybe the first hints of the darker chromosome structure.

dmel-polytene-2014-07-28-11-55-11-cells

The next image is with higher magnification.  You can see the cell nucleus as spheres with darker chromosomes winding around.  In preparing the tissue for these images we apply a lot of pressure to purposely try to rupture the cells and spread the chromosomes (i.e., this is not necessarily what they would normally look like).

dmel-polytene-2014-07-28-12-01-18-nuclei

In the image below one of the nuclei has been squashed and "spread" a bit more than the others.  The chromosomes look like darker squiggles.

dmel-polytene-2014-07-28-12-06-44-nuclei

The image below is zoomed in to an even higher level.  You can start to make out the dark and light banding pattern along the chromosomes.

dmel-polytene-2014-07-28-11-59-22-chromosomes

Below are two nuclei.  The one on the left has been spread more than the one on the right, and the banding pattern is easier to make out---at least for the section that is spread.

dmel-polytene-2014-07-28-12-03-43-chromosomes

In the image below it looks like some of the strands are starting to shear.  However, the end of one chromosome is visible to the right so I wanted to focus on it and see if I could identify which chromosome it belonged to.

dmel-polytene-2014-07-28-12-09-14-spread

Below is zoomed in some more on this area, and this is pushing what the optics in our system are capable of.  Just down from the end of the chromosome is a "puff" where it widens out.  These correspond to areas with active transcription of genes.

dmel-polytene-2014-07-28-12-12-23-spread-detail

There are cytological maps of the chromosomes available at flybase to compare to.  Here is the image at the end of the X-chromosome.

Dmel-X-cyto-end

Based on the similarity in the banding pattern and the presence of the puff I suspect this is the end of the X-chromosome.  The gene yellow (y) which affects body pigment is at the very end and white (w) which results in red eyes when functioning correctly (and happens to be the first genetic variation discovered in Drosophila melanogaster by T. H. Morgan's lab) is just down from the puff at the second dark band.

Ironically the best images today were not from the orcein stained slides but from Hoechst 33258 stain, but strangely the chromosomes did not appear to fluoresce under UV light.  Kenton wrote down all the steps he took today and the most important thing for now is to replicate the process tomorrow to make sure we can get good images of the chromosomes.  Then we will try to fine tune and troubleshoot some more.

Onward to Sydney and then back to Hawai'i

After Melbourne Jolene and I flew to Sydney to attend the GSA annual meeting where both of us gave presentations.  I talked about our plans with underdominance here in Hawai'i and Jolene talked about her work with immunity gene diversity in some island bird species.  After the meeting I returned to Hawai'i but Jolene is traveling on to New Zealand for an invited talk at Otago.

A great day in Melbourne!

We met with Prof. Scott O'Neill and his lab today.  We discussed what we are trying to do in Hawai'i with underdominance and possibilities with Wolbachia in mosquitoes in Hawai'i and Wolbachia strains from Hawaiian Drosophila species.  He is dean of the college and very busy but took time to talk with us and set up a tour and meetings with people working in his lab.  Their main focus is on modifying mosquitoes to block the spread and transmission of Dengue (the virus that causes Dengue fever) by using Wolbachia injections.  Long story short, certain Wolbachia (a type of symbiotic bacteria) strains can infect and be stably transmitted over generations in mosquitoes (and many other insect species).  If at a high enough starting frequency they increase in frequency in local populations and move toward 100% frequency.  Importantly, Wolbachia has also been shown to reduce the transmission of certain disease causing viruses and Plasmodium.  Originally they used a MelPop strain (from Drosophila melanogaster fruit flies) that reduced mosquito lifespan and virus transmission but it reduced fitness of the mosquito too much in the wild and would be lost.  They switched to a Mel strain (also from Drosophila melanogaster) that also (incompletely) blocks transmission and is able to become stably established in the wild.  They are conducting releases of Wolbachia infected mosquitoes in Australia and work in SE Asia.  We also discussed the regulatory aspects of their work both in Australia and internationally.  We got to see how they were raising and working with the mosquitoes, from cages to collecting and feeding to biosecurity setup (most of the work is at level 2 but they have a level 3 lab for the Dengue virus work).  We even got to see them do mosquito (Aedes aegypti) egg micro-injection and Jolene had a shot at arranging the eggs under a microscope and injecting them with a new experimental Wolbachia strain using a micromanipulator.  The people here were extremely helpful and friendly and we got to ask them questions for several hours---we were literally there all day and they took us out to lunch midday.  We have plenty of new pointers to help us with working with the mosquitoes in our lab.  The data they are getting back from the releases are impressive.  They are collecting mosquitoes from the release sites and areas around them weekly and plotting maps of the increase in allele frequency of the Wolbachia infected mosquitoes and the geographic spread from the release range.  On top of this you can see population fluctuations, drift, the effects of rainfall on the wild populations, etc.

A nature walk

I had some free time after the conference ended and before my flight back to Hawai'i---this is very rare for me.  So, I took advantage of the opportunity to go for a nature walk around the UMBC campus.  I saw lots of plants and animals including several deer, rabbits, squirrels, a groundhog, geese, barn swallows, etc.  I also brought along a camera and got photos!  This is a space holder post for now, because I have to get get ready to the airport soon, but I will update with some pictures and commentary as soon as I get a chance.

Synthetic Biology Workshop, Done

The HHMI/NSF sponsored workshop on using synthetic biology as an undergraduate teaching tool has finished.  I am really glad I came to this.  I learned about some impressive new tools, Golden Gate Assembly, GoldenBraid Cloning, C-dogs for uniform gene expression control, reasons to use doubled translation termination sites in plasmid design, The Oligator, new long strand cheap oligo synthesis technologies, just to name a few.  And a lot of good ideas to use both in teaching and research.  We put together 10 minute presentations that are proposals for ways we might use synthetic biology teaching at our home institutions and presented them today.  I teamed up with Mark Wilson of Humboldt State University and we presented something about using Vibrio bacteria as a marine model in synthetic biology.  I have already uploaded a version (edited for clarity---this is not the cleanest presentation I have made but go easy on me---it was put together quickly in a very short amount of time as a part of the workshop) of our presentation to the posted presentations page.

There were also some other impressive presentations.  One that I really liked was a way to use plasmid construction as a random number generator.  Generating random numbers is actually quite difficult to do well using software on a computer, but it may turn out to be easy using the orientation of a promoter with two reporters and a FACs machine (fluorescent activated cell sorter)!  The same group also suggested using a mix of transformed bacteria colonies to simulate genetic drift and/or selection with repeated sampling and plating.  There was another presentation on using E. coli to produce wintergreen to prevent colony collapse disorder (due to mites) in honeybees.    And also a presentation on designing bacteria for rapid, efficient, and specific gun residue detection at crime scenes, etc.

Synthetic Biology Workshop

This week I am on the US East Coast for a GCAT synthetic biology workshop sponsored by NSF and HHMI.  The focus of the workshop is on using synthetic biology as a tool to teach undergraduate genetics research.  Naturally I was speculative about just how "new" synthetic biology is, etc. but I am already impressed by some of the things we went over at our first sessions last night: the Golden Gate plasmid assembly process for example.  Some of the focus of synthetic biology (which is argued to set it apart from genetic engineering in general) does not come naturally to many biologists but is said to be heavily influenced by engineering and computer science, such as standardization and modularity.  There is also a comfort with "black boxes."  You don't have to understand the (generally known) details about how something works to use it (indeed the focus is on using any tool available without a detailed explanation of the mechanism), which does not come easy for many geneticists.  However, they made some convincing arguments and examples of the utility of this approach.  Part of why I am here is to get ideas for undergraduate research and education but I can say I've already seen how this could be useful in plasmid design for some of our other projects.

Also, I am grateful to the conference organizers for breaking some rules to allow me to be here.  The workshop is designed to have teams, preferably a biologist and non-biologist, come from an institute to be trained and take back cross disciplinary ideas with them to their home institute.   I could not find anyone at UH to join my team before the application deadline; so I figured I had nothing to loose and sent in my application anyway with a brief explanation, and was accepted solo!

Frustrating Review Process

At times the peer-review process can be very frustrating.  It is probably wiser not to publish this but I feel like I have to speak out about this somehow---if for no other reason than to illustrate part of the publication (and also grant review) process.  Our two recent publications were in review at various journals for years.  It is painful to see other publications come out that are similar to what you have been trying to publish and to see so much time going by on the calendar during the process.  The peer review process is necessary, and on the balance helps improve the quality of scientific publications.  I can honestly say that some of my publications have greatly benefited from reviewer feedback.  However, this is not always the case.  I think removing the authors names and institutions from the material the reviewer sees, among other steps, would improve the process.  At any rate, I have saved the following review as an example of what we went through to get our papers published (this one is from a previous submission of the engineering underdominance manuscript).  This reviewer followed our manuscript submission from one journal to another (the same person was picked repeatedly by the editors).  We could tell this because some of the reviews contained identical word-for-word copy and paste sections despite our revisions to try to address and clarify them.  I have added the emphasis but the words are taken directly from the review.  If you read through to the end you can see that the reviewer made up speculative scenarios that were not in our paper as a basis to reject it.


August 21, 2012

Unfortunately, the authors have failed to generate the underdominance-based gene drive system they claim to have created, and thus the paper must be rejected. In fact, their text is written in such away that the fact that they have not in fact created a functioning gene drive system only becomes clear when one reads the methods.

In brief, their strategy is to use RNAi, with the dsRNA generated targeting a ribosomal protein, and being expressed under the control of a gal4 uas element. Flies carry this piece of DNA and in addition a rescuing version of the ribosomal protein that is resistant to the dsRNA-dependent RNAi. Flies carrying this chromosome (the third chromosome) are argued as showing gene drive (below). But of course this is nonsense. This chromosome does nothing at all in a wildtype population. Absolutely nothing at all. It does not drive. It does nothing but float in a wildtype population.

The only context in which the transgene-bearing third chromosome does show drive involves a trick, hidden in the methods. The authors take their rnai-rescue line and introduce into this genetic background a transgene on the second chromosome bearing an actin promoter driven version of GAL4, which drives transcription from UAS elements. Thus, in this new strain, acting gal4/Cyo balancer; uas rnai-rescue, we now get effects. The actin-GAL4-driven dsRNA knocks down the expression of the ribosomal protein; in the presence of one copy of the rescue construct (in heterozygotes) they are less fit, and show a lower viability. Very importantly for the authors technique, they are not dead. Otherwise they would never have been able to cross them with each other to generate homozygotes. In homozygotes for the third (The second chromosome balancer prevents GAL4 from ever becoming homozygous), the animals are more healthy than heterozygotes (but of course only heterozygotes that carry a gal4-bearing second chromosome), but less fit than wildtype.

This genetic behavior allows them to show a form of underdominance - in an extremely contrived and artificial situation - that fairly boggles the mind. They take their actin-gal4/cyo; rnai-rescue flies and cross them to their "wildtype" strain, which is balanced on the second chromosome for actin-gal4/cyo but wildtype for the third chromosome. In other words, their wildtype strain always contains actin-gal4, but not in their actual construct. In the text the authors say that the use of gal4 is not essential for their system and is only meant to be illustrative. But this is nonsense. They simply cannot generate drive without the presence of a gal4 that is introduced into their genetic background.

Not surprisingly, in the above, contrived situation the authors can show underdominant behavior. When the frequency of the rnai-rescue chromosome reaches some frequency that depends on the relative fitness costs associated with being heterozygous and homozygous for the chromosome (but only in the presence of a actin-gal4, the benefits of being homozygous for the rnai-rescue chromosome outweigh the benefits of carrying a wildtype chromosome (which at this point finds itself in unfit heterozygotes most of the time) and the uas-rnai-rescue chromosome drives to allele fixation. Below this threshold the chromosome disappears from the population.

Again, the paper must be rejected because they have simply not created a driving underdominant chromosome that can drive itself into a wildtype population. It is remarkable that the authors believe that they can get away without generating a simple three gene construct in a model organism such as drosophila, that would show drive.

In thinking about why the authors might not have done such an obvious and essential experiment one can only conclude that they probably tried but were unable to generate the relevant transformant. There is probably a very good reason for this, and it relates to the reason why no underdominant gene drive systems have ever been created using modern genetic engineering techniques: It is very hard (if not impossible) to generate the initial unfit heterozygote, which will ultimately give rise to the more fit homozygote. Unfortunately, without a demonstrated solution to this problem all engineered underdominance systems are so much hot air. They look good on paper, but in fact cannot be generated in actual living organisms.

And our Medea-Underdominance manuscript is also out in print!

GokhaleEtAl2014

Right on the heels of the last paper our theory results on a combined Medea-Underdominant dynamics is finally published.  Like the last one this took several years going from journal to journal and through a long line of reviewers before finally getting published.  Chaitanya really deserves the credit on this one for making the final push to resubmit yet again and get it out there.

Our population genetic engineering paper is published!

reevesetal2014

At long last, our paper describing engineering underdominance in Drosophila with haploinsufficient poison/rescue is out!  Here is a link to the full article.

In other news, classes and committee work are finally done, so I can have a chance to get back into the lab.  My preproposal to NSF was turned down again but a preproposal to NASA has made it to the next stage to submit a full proposal!