Exploding Cars and Beautiful People May 26, 2000

Well, it looks like we have really made the big time (By we, I mean our industry). The latest Mission: Impossible movie uses biotechnology as the McGuffin. This is a term coined by Alfred Hitchcock. It stands for the plot device that gets the movie's action going. It can be anything and 'what it is' is not as important as the fact that it provides a need for our protagonists to act. In MI2, the McGuffin is a genetically engineered influenza virus, developed by a small lab in Australia (I was hoping it would be New Zealand). It kills its victims in an incredibly short time if they do not get the antidote. The bug, and its antidote, is prevented from arriving at the CDC by a rogue IMF agent, and so the story goes... (Of course, this makes TWO missions in a row in which the IMF has done a really awful job choosing its agents for a mission. Oh, for the days of Peter Graves and the Real IMF.)

The biotech company wants to use the super virus as a niche for its antidote. See, all those greedy companies want to do is make us sick first and then cure us. This fits right in with some of the real paranoid literature out there (Or with an X-files episode). But it is nice to see that biotech companies are on the radar screen of Hollywood. Now the rogue agent is just nuts. The real laughable part of the movie is that the rogue agent does not really want money. He wants stock options (!!) in the biotech company that makes the virus. Figures it will go through the roof when he releases the virus into the population of Sydney. Yeah, stock options. That's real good. Doesn't he read the paper. The last time I saw an evil mastermind make such a foolish error was Dr. Evil asking for '1 milllllion dollars' in Austin Powers.

Of course, the biology in this movie is pretty laughable (i.e. the entire breeding stock of virus is kept in 3 petri dishes; DNA sequencing of the virus takes about 5 seconds) but the details really do not matter. It is a John Woo movie and it shows. Slow motion ballets with bullets, two fisted automatics, white doves in super slow motion and beautiful people driving motorized vehicles at high speed, blowing up lots of extraneous cars. I loved it. Just wish there had been more. (Plus there just was not enough of Ving Rhames.)

Enough on biotechnology in the popular media. I am going to continue talking about some popular biotechnology in the scientific media. Okay, I promise, this will be the last column for awhile about retrotransposons and such. But there have just been such a wealth of information recently, most likely due to the ongoing sequencing projects, that I had to include a few more. But this week's column deals with more than just transposable elements hopping around the genome. It discusses actual genes and the altered forms they take BECAUSE of retrotransposition. These are not some theoretical proposals regarding the effects of LINE sequences and their ability to remake the genome. These are real effects, seen in humans.

Earlier, I mentioned processed pseudogenes, the passive byproduct of LINE retrotransposition. This work used engineered sequences to create a processed pseudogene, demonstrating the basic principles. But there is a critical difference between a processed pseudogene and a retrotransposon. The same enzymatic process (i.e. reverse transcriptase and endonuclease) that takes the LINE mRNA sequences and place them back into the chromosome can be applied to any mRNA. However, the LINE sequence often recreates a polII site for itself. A processed mRNA can not do this, and will be inactive, fit only for eventual decay into oblivion, when inserted.

Unless it somehow places itself next to a promoter. This could allow it to be expressed and continue to be functional. This situation has been described. A large portion of the Y chromosome does not recombine with any portion of the X chromosome (called NRY - non-recombining region of the Y chromosome). These NRY regions are Y specific and thus, only found in the male. For over 40 years, this region was believed to be useless, a wasteland of decaying, inactive genes and repetitive DNA. Recent work has done much to dispel this view. There are several gene families in this region that are not only active but important.

CDY, standing for chromodomain protein on the Y chromosome (great jargon), produces mRNA that is only found in the testis, not in any other tissue. The genomic structure of this gene was determined and, surprise, was found to be exactly colinear with the mRNA. That is, it had no introns and it appeared that a poly-A tract was placed immediately adjacent to the stop codon, much, much closer than any other nuclear gene. All the hallmarks of a processed pseudogene, except that it is an actual , functional gene, no pseudo about it.

So, if CDY is a processed gene, where is the gene whose mRNA CDY is derived from? Examining Northern blots at low stringencies provided the answer. Mice have no CDY gene present on the Y chromosome, but a gene called Cdyl, that has about 63% homology with CDY, was found on an autosome. It has introns (seven of them), and looks like a normal gene.

It turns out that there is a human homologue to Cdyl. This gene, CDYL, has 93% homology to the mouse gene and shares almost all the same intron-exon boundaries as Cdyl. The murine and human genes are syntenic, being found in similar chromosome locations. So, we have 2 human genes, CDYL and CDY. CDYL and the mouse gene Cdyl are homologues and are expressed in many different cell types in both organisms. But, interestingly, in mice there is a second transcript from Cdyl of the exact same size as the human CDY transcript and it is ONLY expressed in testis. So in mice, there is 1 gene but it is processed in 2 different ways in different cells. One form of processing is testis-specific. In humans, there is more than 1 gene and they are expressed in 2 different ways indifferent cells. One form of expression is testis-specific. Kind of neat, how cells are able to accomplish the same mission but use two different processes, one of which is due to retrotransposons.

So, there was an ancestral gene, Cdyl, that was expressed and spliced in 2 forms, one of these though was only found in the testis. Sometime about 40-50 million years ago (as determined by looking at other primates) something happened and the testis-specific transcript was placed in a processed form on the Y chromosome. Now the original gene no longer expesses itself in any alternative form in humans, since there is now a gene elsewhere that accomplishes that purpose. Makes for an interesting story and a novel way to create a second gene, rather than simply relying on differential splicing.

Now, while doing some interesting and somewhat novel literature searching last week (a potential topic for a future column) I ran across an article that really demonstrates the ability of LINEs to affect alternative splicing. Above, I have talked about the indirect effect of LINES to change alternative splicing pathways that were already taking place. Now, I will discuss how LINEs can actually create alternative splicing. This is from the most recent edition of PNAS.

Some background. Attractin is a soluble human protein with an enzymatic activity (dipeptidyl peptidase IV) similar to CD26. It is found on activated T cells. A murine homologue of the gene, mahogany, has been described, but it is obviously membrane bound. Is there a membrane-bound form of human attractin? Is it a separate gene from soluble attractin? So, to investigate the possibilities, the genomic sequences of the human attractin were investigated. And, sure enough, there is a larger length transcript that produced a membrane-bound form of the human protein. And it is generated from the same sequences as the soluble form, just by an alternative splicing pathway.

There are 30 exons in the genomic sequences of attractin. Exon 25 is normally skipped in order to create the transmembrane version. The soluble form is created when exon 25 is included. Exon 25 contains a stop codon and a genomically encoded poly-A tract 196 base pairs downstream from it. The 196 bases correspond exactly with the 3' end of ORF 2 of LINE-1. The leftover remnant of a retrotransposition event. Although the corresponding area of the murine genome has not been investigated, there is circumstantial evidence to indicate that the insertion occurred recently.

So, a retrotransposon inserted itself into a genomic sequence, providing the molecule in question the ability to be secreted into the serum or remain tethered to the cell membrane. It did not passively move a processed gene into the genome or disrupt an existing gene. It did more than just affect the expression of a gene. It actually altered the structure of the gene's protein product, creating a totally novel form of the protein. This sort of genetic manipulation presents incredible possibilities, particularly with the propensity of these transposable elements to carry along 3' non-coding regions. Junk DNA may be incredibly important for rapidly altering the landscape of a genome, rather than just decaying fossils of earlier times.

Many species do not have many retrotransposons or much junk DNA. Perhaps this is a relatively recent occurrence. The testis specific retrotransposition happened roughly 40-50 million years ago. This is about the same time that the B-LINE sequences entered ruminates from reptiles. Could there have been some sort of world wide infection of species by retrotransposons at this time? Could much of the evolution of modern animals be tied to this event? Wow, sounds like a great idea for a movie or book. I just have to think of a way to get exploding cars being driven by beautiful people in it.