fied more than 23 

 different stages in 

 the organism's life 

 cycle, and most 

 resemble other 

 common, nontoxic 

 dinoflagellates or 

 even the sediments 

 in which they lie. 



So how do 

 scientists know 

 whether Pfiesteria 

 inhabits a particular 

 river? The simple 

 approach is to test 

 water from that 

 river. A test for 

 Pfiesteria, however, 

 is not so simple. 



Currently, 

 samples of water 

 thought to harbor 

 the dinoflagellate 

 must be transported 

 to a laboratory and 

 the creature 

 identified visually 

 by researchers or 

 technicians familiar 

 with it. Even that 

 can be a difficult 

 task because the 

 organism looks very 

 similar to other 

 dinoflagellates. A 

 reliable test, then, 

 would be a valuable 

 tool in tracking 

 down Pfiesteria. 



The probe that 

 Rublee and his 

 graduate students are developing 

 could be that tool. 



ft 



3P IS 



Studying an autoradiograph of a sequencing gel 

 to examine parts of a Pfiesteria gene that nas been sequenced 



A Tough Nut to Crack 



Fish kills seem worlds away from 

 Rublee' s office and laboratories on the 

 UNC-Greensboro campus. From his 

 cramped space lit by windows high 

 above his desk, Rublee plans his work 

 amid filing cabinets and shelves piled 

 with books, papers and notebooks. 

 The small laboratory just outside his 

 office door holds a bewildering array 



of all the standard scientific accoutre- 

 ments: beakers, test tubes, plastic vials 

 and canisters, capped bottles, micro- 

 scopes. 



The world of the dinoflagellate is 

 under intense scrutiny here, and that 

 work is anything but standard. The 

 many unknowns about Pfiesteria have 

 made the research almost maddening 

 at times for Rublee and his cohorts, 

 particularly his former graduate 

 student Kristen Toffer. 



"Kristen worked a long time 



learning how to deal 

 with the organism. 

 We thought we 

 were doing the right 

 things, but we 

 weren't getting very 

 far, and that was 

 frustrating," Rublee 

 says. "It wasn't 

 kick-the-wall 

 frustration but darn- 

 it-this-should-have- 

 worked frustration. 

 You wonder, so 

 what do you do 

 next? If it doesn't 

 work, you step back 

 and analyze why it 

 didn't and ask, 

 should it have 

 worked?" 



That approach 

 is taken by count- 

 less researchers 

 around the world 

 every day, and 

 Rublee and his team 

 have become 

 familiar with it. 

 Pfiesteria, the 

 scientist says, is "a 

 tough nut to crack." 



The nut Rublee 

 is cracking is 

 Pfiesteria 's genetic 

 code — a successful 

 probe depends on it. 



To decipher the 

 code, Rublee 

 removes the DNA 

 from a Pfiesteria 

 gene and replicates a long portion of it 

 through a process called polymerase 

 chain reaction, which Rublee describes 

 as DNA replication in a test tube rather 

 than a cell. Simply put, the DNA 

 double helix is heated, breaking and 

 separating the bonds of the single 

 strand. Rublee then adds primers, 

 synthesized pieces of DNA that match 

 up with segments called conserved 

 regions. These sections of a gene are 

 virtually identical for all organisms and 

 thus are well-known. 



6 MAY/JUNE 1997 



