At last, a Russian-American research group has found a use for junkDNA. With it, they hope to transform current methods of isolatingand cloning human genes.

A good 20 to 25 percent of a chromosome's nucleic acid consists ofrepetitive stretches, which show no signs of gainful employment.Now, molecular geneticist Michael Resnick and cell biologistViadimir Larionov are perfecting an approach to simplify, sharpenand speed up the identification of desired gene families, andeventually of individual single-gene sequences.

They presented a report on their system early last month in theProceedings of the National Academy of Sciences (PNAS), datedJan. 2, 1996. Its title: "Specific cloning of human DNA as yeastartificial chromosomes [YAC] by transformation-associatedrecombination [TAR]."

Resnick heads the chromosome stability group at the NationalInstitute of Environmental Health Sciences (NIEHS), in ResearchTriangle Park, N.C. Larionov and his fellow Russian scientist NatalyaKouprina, are on leave from the Institute of Cytology in St.Petersburg, Russia.

TAR, Resnick told BioWorld Today, is based on baiting the gene-fishing hook with DNA repeats. Here's how:

"Our basic idea," he explained, "is to take human cells and gentlyisolate their total genomic DNA. This consists 20 to 30 percent oflarge repeats, spaced 2,000 to 3,000 bases apart. Then we hook upthese chromosomal sequences to opened plasmids, with ends thatterminate in repeat sequences largely homologous to those in ourDNA of interest."

The cells of baker's yeast, Saccharomyces cerevisiae, Resnickobserved, "are pretty efficient at taking up vector DNA along withone or a few large pieces of human chromosomal DNA. Once sittinginside a yeast cell," he continued, "the newly transformed nucleicacid will efficiently undergo recombination. The repeats on thevector and on the chromosomal DNA will find each other and join bycovalent linkage. This generates an artificial chromosome not unlikethe yeast cell's own chromosome."

Resnick contrasts this technique to current methodology: "At present,there is no specific way of getting a specific gene. Right now, onemust clone randomly, cutting the DNA from cells into thousands ormillions of fragments. After cloning into yeast or bacteria, each clonehas to be examined to identify the DNA of interest, usinghybridization probes."

"The technique that TAR contributes to this process," Resnickcontinued, "is the concept of transforming with total DNA, andhaving all the reaction in vivo inside the cell. The yeast is doing thework."

That doesn't mean the NIEHS team is standing by idly. Sincesubmitting their paper to PNAS, Resnick and his co-authors cite"several advancements" they have made en route from research anddevelopment toward clinical application. They reported these at aDepartment of Energy genome science meeting on Jan. 31, 1996, inSanta Fe, N.M.:

* "We demonstrated we could use TAR for specific cloning of atleast one family of genes, the human ribosomal genes," Resnick said.

* "In our original paper," Larionov observed, "the yield of humanpositive clones approximated 20 to 25 percent. Now we are able toselect and isolate human DNA from chromosomal hybrids at close to100 percent.

* "Also, it's a general desire to isolate pieces of human DNA in amouse background, for positional cloning. Mouse DNA has norelated repeats. If we had done just random cloning," he pointed out,"about 98 percent of the yeast artificial chromosomes would containmurine DNA, and 2 percent human. We turn that around, and find 60to 70 percent human, and about 30 percent mouse."

* To which Resnick added that cloners working in the HumanGenome Mapping Project are plagued by mouse-human chimeras,whereas with TAR, "there are no chimeras formed that contain bothhuman and mouse DNA."

The team is actively pursuing a gene that expresses the Ku protein,which is important in repairing DNA double-stranded breaks inhuman cells. It aims to clone Ku's promoter.

"Our plan," Resnick said, "is to go after entire DNA repair genes, andobtain additional clones relating to the breast-cancer genes, which arebeing pursued at this institute. (See BioWorld Today, Sept. 19, 1994,p. 1.)

Mutations involved in such diseases, Resnick observed, "can bestudied relatively easily right now in coding sequences. Those inpromoter regions or introns are not readily accessible by currentmethods. They might be by TAR."

NIEHS has decided not to patent the TAR procedure, Resnick said. n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.