lecular, Cellular, and Developmental Biology at 
the University of Colorado at Boulder and Profes- 
sor of Biochemistry, Biophysics, and Genetics 
at the University of Colorado Health Sciences 
Center, Denver. 
Articles 
Cech, T.R. 1992. Ribozyme engineering. Curr 
Opin Struct Biol 2:605-609. 
Davila-Aponte, J.A., Huss, V.A.R., Sogin, M.L., and 
Cech, T.R. 1 99 1 ■ A self-splicing group I intron in 
the nuclear pre-rRNA of the green alga, Ankistro- 
desmus stipitatus. Nucleic Acids Res 19:4429- 
4436. 
Fang, G.W., and Cech, T.R. 1991. Molecular clon- 
ing of telomere-binding protein genes from Sty- 
lonychia my tills. Nucleic Acids Res 19:5515- 
5518. 
Gampel, A., and Cech, T.R. 1991. Binding of the 
CBP2 protein to a yeast mitochondrial group 1 in- 
tron requires the catalytic core of the UNA. Genes 
Dev 5:1870-1880. 
Gray, J. T., Celander, D.W., Price, CM., and Cech, 
T.R. 1991. Cloning and expression of genes for 
the Oxytricha telomere-binding protein: specific 
subunit interactions in the telomeric complex. 
Cell 67:807-814. 
Heuer, T.S., Chandry, P.S., Belfort, M., Celander, 
D.W., and Cech, T.R. 1991. Folding of group I 
introns from bacteriophage T4 involves internal- 
ization of the catalytic core. Proc Natl Acad Sci 
USA 88:11105-11109- 
Piccirilli, J.A., McConnell, T.S., Zaug, A.J., NoUer, 
H.F., and Cech, T.R. 1992. Aminoacyl esterase 
activity of the Tetrahymena ribozyme. Science 
256:1420-1424. 
Pyle, A.M., Murphy, F.L., and Cech, T.R. 1992. RNA 
substrate binding site in the catalytic core of the 
Tetrahymena ribozyme. Nature 358:123-128. 
Wang, J.-F., and Cech, T.R. 1992. Tertiary struc- 
ture around the guanosine-binding site of the Tet- 
rahymena ribozyme. Science 256:526-529. 
Young, B., Herschlag, D., and Cech, T.R. 1991. Mu- 
tations in a nonconserved sequence of the tetrahy- 
mena ribozyme increase activity and specificity. 
Ce// 67:1007-1019. 
GENOMIC SEQUENCE COMPARISONS 
George M. Church, Ph.D., Assistant Investigator 
Several laboratories are sequencing small ge- 
nomes (1-100 Mbp) from each of the three phylo- 
genetic kingdoms. Comparisons of these sequences 
will define consensus sequences for most classes of 
protein domains, evolutionary conservation, and 
change. Up to 20-fold higher substitution rates in 
nonconserved compared with coding nucleotides 
allow the discrimination of random open reading 
frames (ORFs) from those encoding proteins that 
confer a selective edge. The exceptionally extensive 
genetic maps, biochemical pathways, and regula- 
tory landmarks for these organisms reveal physiolog- 
ical relationships and clues to functions for new 
sequence elements. The genome closest to comple- 
tion is Escherichia coli, with more than 40% of its 
4.7 Mbp completed by the work of 2,000 scientists. 
Dr. Church's laboratory is focusing on developing 
technology of general use in genome-sequencing 
projects, with emphasis on ways to improve accu- 
racy and biological interpretability. 
DNA Sequencing and Automation 
Multiplexing is a general method of mixing and 
separating informational packets. In multiplex se- 
quencing, pools of up to 40 strategically tagged 
DNA samples flow as if one throughout most of the 
protocol steps. Direct-transfer electrophoresis modi- 
fied for multiplex and duplex sequencing yields 
runs with error rates <0.06% for the first 400 bases 
and <0.6% for the first 800 bases. The new chemilu- 
minescent substrate CSPD yields three times faster 
exposures — in the range of 10 to 60 min. An auto- 
mated PC-controlled drum device now handles the 
hybridization, washing, and substrate addition steps. 
Dr. Church's laboratory has recently integrated di- 
rect-transfer electrophoresis, automated multiplex 
hybridizations, and automated film reading to se- 
quence three E. co// cosmids. Sequence patterns for 
two cosmids were detected using chemilumines- 
cence with oligonucleotide probes directly conju- 
gated to alkaline phosphatase. Primers for the di- 
rected walking and dideoxy sequence confirmation 
steps contain 1 5 base tags for detection. For the cos- 
mids, 20 gels resulted in 9,216 sequences on film. 
The programs REPLICA and GTAC automatically 
read and assemble the data from films. Another pro- 
gram automatically finds and graphically annotates 
ORFs, including matches to database sequences. 
GENETICS 1 69 
