G. RF2 gene ofE. coli (Liming Young). Overexpres- 
sion of RF2 (ribosome release factor) inhibited the 
growth of E. coli. Deletion of the carboxyl terminus 
(—70 aa) inactivated RF2 activity and allowed nor- 
mal growth when overexpressed. Further genetic 
experiments are aimed at defining the crucial re- 
gion. RF2 protein has five leucine repeats in its 
amino terminus. Whether it is a leucine-zipper re- 
peat requires further research. 
II. Genome Structure. 
A. Bends in DNA (Dr. Debra Milton and Mark Cas- 
per). Saturation mutagenesis of a 60 bp bend re- 
gion of a naturally occurring SV40 DNA fragment 
has identified bases that influence the DNA bend. 
As expected from current models, interruption of 
an A tract or an alteration in the A tract phasing de- 
creased the bend. However, G tracts influence a 
DNA bend when spaced at the half-period between 
periodically phased A tracts. Also, ApG or GpA di- 
nucleotides are influential. Clearly, base pairs other 
than dA-dT are involved in influencing the DNA 
bend and provide a good database for further DNA 
model building. 
B. Organization of maize mitochondrial genome 
(Dr. Christiane Fauron and Marie Havlik) . In maize, 
the physical map of the normal mitochondrial DNA 
(N) has a sequence complexity of 570 kb, com- 
pared with 540 kb for the cytoplasmic male sterile 
type Texas (cmsT) . For both genomes the entire se- 
quence complexity can be represented on a master 
chromosome or as a multipartite structure by 
recombination at repeated sequences. A more de- 
tailed comparison of various N and cmsT mitochon- 
drial genomes revealed some rare microhetero- 
geneity (e.g., point mutations, small deletions/ 
additions creating different restriction sites) be- 
tween some N cytoplasms. The mitochondrial ge- 
nome from two fertile revertants, V3 and V18, de- 
rived from a cmsT callus tissue culture has been 
determined. The sequence can be mapped onto cir- 
cular "master chromosomes" of 705 kb and 818 kb, 
which include one large duplication and one small 
deletion. As found for the maize N type and cmsT 
mitochondrial genomes, the V3 and VIS master 
chromosomes also exist in a multipartite structure 
generated by recombination through repeated se- 
quences. 
III. DNA Sequencing Technology. 
A. Large-scale sequencing (Dr. Weiss and D. Dunn). 
Attempts to sequence cosmid DNA efficiently are 
continuing. Two cosmids containing human geno- 
mic DNA from the neurofibromatosis (type 1) re- 
gion of chromosome 17 have been randomly sub- 
cloned and sequenced using a multiplex protocol. 
This experiment allowed rapid accumulation of 
85% of the sequence in a random fashion. Analysis 
of the pace of this particular multiplex strategy has 
impelled the implementation of a new subcloning 
strategy employing multiplexed self-replicating 
transposons. This protocol will simplify and accel- 
erate the rapid accumulation of sequence data. 
As part of this laboratory's participation in the 
Human Genome Project, several other avenues 
for improving DNA sequencing technology are 
being pursued. An optics laboratory for lumines- 
cent DNA sequencing has been assembled by Jeff 
Ives and Dr. Achim Karger to assess fluorescent and 
chemiluminescent substrates to probe DNA se- 
quences on polymer membranes. The membranes 
are then imaged on a cryogenically cooled charge- 
coupled device (CCD) array. The possibility of DNA 
fractionation for sequencing using microbore ( —74 
|xm) capillaries is being tested by Harold Swerdlow. 
Heat dissipation and power requirements are re- 
duced substantially in capillaries, which permits 
gels to be electrophoresed at voltages 10 to 15 
times higher than for conventional separations, 
thus increasing resolving power. Mike Murdock is 
progressing on development of an autoradiogram 
reader, using an artificial neural network that is 
trained on 5,000 sample images to iteratively evolve a 
set of network weights that are then used to read ar- 
bitrary gel images. 
Dr. Gesteland is also Professor of Human Genet- 
ics and of Biology at the University of Utah School 
of Medicine. 
PUBLICATIONS 
Articles 
Falahee, M.B., Weiss, R.B. , O'Connor, M., Doonan, S., Gesteland, R.F. , and Atkins, J.F. 1988. Mutants of trans- 
lational components that alter reading frame by two steps forward or one step back. / Biol Chem 
263:18099-18103. 
Continued 
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