Laboratory
Procedures
Introduction
Scientific Writing Resources
Link to Electronic-version of the Science journal articles to be used
in graded in-lab discussions
Links to Information Applicable to Lab Series:
 Series I: Identifying Mutations in a Transcriptional Regulator, Lambda
Repressor Protein
Series II: Suppressing a Cell Cycle Control Mutant in Yeast
Series III: Plant Genetic Engineering
Introduction
to the Genetics Lab [back to top]
The lab portion of the genetics course at Wellesley College is designed
to introduce you to many of the common tools and techniques used in investigation
of biological processes through study of genes or gene products. The
lab is designed to model inquiry based hypothesis driven research, very
similar to the way you would approach biological questions in a real
research lab. We will investigate gene regulation at the level of transcription
in a prokaryotic system in a month long investigation of lambda repressor
protein in E. coli bacteria. We will then turn our attention to a simple
eukaryote in a three weeks investigation of cell cycle control genes
in yeast. We will complete our semester with the study of transgenic
tobacco plants that you have genetically engineered to express a bacterial
gene and then nurtured to maturity over the semester. The lab uses sophisticated
materials and equipment and provides invaluable experience in current
molecular and cell biology research technology. An additional goal is
for you to learn scientific writing in preparation for publishing your
future research in a peer-reviewed scientific journal. We hope that you
will enjoy the lab portion of the course as much as we enjoy teaching
it.
The Lab Instructors of BISC 219
Scientific
Writing Resources [back to top]
The Student Research Survival Kit
The Research and Instruction Librarians
at Wellesley College (with much thanks to Suzanne Alcott) have put
together a web
page to help students with the mechanics (as opposed to content)
of paper-writing.
Other web resources that are particularly applicable to science writing
are:
- How
to Write a Paper in Scientific Style and Journal Style and Format
The citation style mentioned at this site is most closely applicable
for biologists, although specific disciplines in biology may not follow
this style completely. Most remarks about writing are on target for all
of the basic sciences.
- Style
Guide on the Web
For BISC 219 papers,
use this CBE (Council of Biology Editors)
site with information about how to cite material
accessed electronically
and from web sources
using
the name-year format option. The site guidelines for citing Internet
sources stem from the principles presented in the sixth edition of
Scientific Style and Format:
The CBE Manual for Authors, Editors, and Publishers, published by
the Council of Biology Editors (now the Council of Science Editors)
in
1994. Please
use
the citation style recommended in the CBE Manual, which
also gives advice
for styling and formatting scientific papers, journals, and books
for publication. Its editors offer two methods for citing and documenting
sources: the citation-sequence
system and the name-year system. In BISC 219, we will require the later.
Links
to the Science journal articles that we will discuss at the end of
the
yeast and plant lab series (available from library e-resources) [back
to top]
- Galitski,
T., Saldanha, A.J., Styles, C.A., Lander, E.S., and G.R. Fink. "Ploidy
Regulation of Gene Expression [pdf]" Science (1999)
285: 251-254.
- Apse,
G.P., Aharon, G.S., Snedden, W.A., Blumwald, E. "Salt
Tolerance Conferred by Overexpression of a Vacuolar Na+/H+ Antiport
in Arabidopsis [pdf]"Science (1999)
285: 1256-1258.
Series
I: Identifying Mutations in a Transcriptional Regulator, Lambda Repressor
Protein [back to top]
- The
DNA Binding Arm of λ Repressor: Critical Contacts from a Flexible
Region
Neil D. Clarke; Lesa J. Beamer; Harry R. Goldberg; Carol Berkower; Carol
O. Pabo
Science, New Series, Vol. 254, No. 5029, Genome Issue: Maps and Database.
(Oct. 11, 1991), pp. 267-270.
Abstract: Segments of protein that do not
adopt a well-ordered conformation in the absence of DNA can still
contribute to site-specific
recognition
of DNA. The first six residues (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6-)
of phage α repressor are flexible but are important for
site-specific binding. Low-temperature x-ray crystallography and
codondirected saturation
mutagenesis were used to study the role of this segment. All of
the functional sequences have the form [X]1-[X]2-[Lys
or Arg]3-[Lys]4-[Lys or Arg]5-[X]6.
A high-resolution (1.8 angstrom) crystal structure shows that Lys3 and
Lys4 each
make multiple hydrogen bonds with guanines and that L ss
interacts with the phosphate backbone. The symmetry of the complex
breaks down near the center of the site, and these results suggest
a revision
in the traditional alignment of the six α operator
sites.
- A
comparative three-dimensional model of the carboxy-terminal
domain of the lambda repressor and its use to build intact repressor
tetramer models bound to adjacent operator
sites
Rajagopal Chattopadhyaya and Kaushik Ghosh
Journal of Structural Biology Vol. 141 (2003) pp.103-114.
Abstract: A model for
residues 93–236 of the λ repressor (1gfx) was
predicted, based on the UmuD' crystal structure, as part of four
intact repressor molecules bound to two adjacent operator sites.
The structure
of region 136–230 in 1gfx was found to be nearly identical
to the independently determined crystal structure of the 132–236
fragment, 1f39, released later by the PDB. Later, two more tetrameric
models of
the λ repressor tetramer bound to two adjacent operator sites were
constructed by us; in one of these, 1j5g, the N-domain and C-domain
coordinates
and hence monomer-monomer and dimer-dimer interactions are almost
the same
as in 1gfx, but the structure of the linker region is partly based
on the linker region of the LexA dimer in 1jhe; in the other, 1lwq,
the
crystalline tetramer for region 140–236 has been coopted
from the crystal structure deposited in 1kca, the operator DNA
and N-domain
coordinates
of which are same as those in 1gfx and 1j5g, but the linker region
is partly based on the LexA dimer structures 1jhe and 1jhh. Monomer-monomer
interactions at the same operator site are stabilized by exposed
hydrophobic side chains in ß-strands while cooperative interactions
are mostly confined
to ß6 and some adjacent residues in both 1gfx and 1j5g. Mutational
data, existence of a twofold axis relating two C-domains within
a dimer,
and
minimization of DNA distortion between adjacent operator sites
allow us to roughly position the C-domain with respect to the N-domain
for
both 1gfx and 1j5g. The study correlates these models with functional,
biochemical, biophysical, and immunological data on the repressor
in the literature. The oligomerization mode observed in the crystal
structure
of 132–236 may not exist in the intact repressor bound to
the operator since it is shown to contradict several published
biochemical
data on
the intact repressor.
- Protein-DNA Recognition:
New Perspectives and Underlying Themes
Peter H. von Hippel
Science, New Series, Vol. 263, No. 5148. (Feb. 11, 1994), pp. 769-770.
Series
II: Suppressing a Cell Cycle Control Mutant in Yeast [back
to top]
Sacchromyces cerevisiae genome data
base (SGD)
This site might be helpful in understanding DNA
micro-arrays.
Links to journal articles:
- High
midsummer for small GTPases [pdf], Trends in
Cell Biology, Volume 12, Issue 11, 1 November 2002, Pages 495-497
Margareta Nikolic and Jonathan Chernoff
- Bud-site selection and cell polarity in budding
yeast [pdf], Current Opinion
in Microbiology, Volume 5, Issue 2, 1 April 2002, Pages 179-186
Antonio Casamayor and Michael Snyder
- Small
G-protein networks: Their crosstalk and signal cascades [pdf], Cellular
Signaling, Volume 12, Issue 8, August 2000, Pages 515-524
Takashi Matozaki, Hiroyuki Nakanishi and Yoshimi Takai
Series
III: Plant Genetic Engineering [back to top]
Boston Globe, Feb. 5, 2002
Scientists focus on the tobacco plant as a possible cancer-fighter
Genetically altered plants may someday produce drugs to combat many diseases
Boston
Globe Nov. 14, 2003 - story about artificial genome construction
Fast method to build genes found
Complete biological systems envisioned
Links to research articles (with abstracts) using gusA gene in a GUS
reporter system:
- Appl. Environ. Microbiol., Jun 1993, 1767-1773, Vol 59,
No. 6
Copyright © 1993, American Society for Microbiology
The GUS gene fusion system (Escherichia coli beta-D-glucuronidase gene),
a useful tool in studies of root colonization by Fusarium oxysporum
Y Couteaudier, MJ Daboussi, A Eparvier, T Langin and J Orcival
Institut de Genetique et Microbiologie, Universite Paris-Sud, Orsay,
France
Abstract: The plant-pathogenic fungus Fusarium oxysporum was successfully
transformed with the beta-D-glucuronidase gene from Escherichia coli
(gusA) (GUS
system) in combination with the gene for nitrate reductase (niaD)
as the selectable marker. The frequency of cotransformation, as determined
by GUS expression on plates containing medium supplemented with 5-bromo-4-chloro-3-indolyl
glucuronide (GUS+), was very high (up to 75%). Southern hybridization
analyses of GUS+ transformants revealed that single or multiple copies
of the gusA gene were integrated into the genomes. High levels of
GUS
activity are expressed in some transformants, but activity in F.
oxysporum does not appear to be correlated with the copy number of
the gusA gene.
Since the highest activity was found in a transformant with a single
copy, it can be assumed that sequence elements of F. oxysporum integrated
upstream of the gene can act as a promoter or enhancer. Expression
of the gusA gene was also detected during growth of the fungus in plants,
indicating that the GUS system can be used as a sensitive and easy
reporter
gene assay in F. oxysporum.
- Development, Vol 112, Issue 4 1009-1019
Copyright © 1991
by Company of Biologists
Functional tagging of regulatory elements in the plant genome
JF Topping, W Wei and K Lindsey
Leicester Biocentre, University of Leicester, UK.
Abstract: In comparison with animals,
relatively few plant genes have been identified that have been shown
to be under organ-, tissue- or cell-type-specific
regulation. In this paper, we describe how the beta-glucuronidase
(GUS) reporter gene (gusA or uidA), fused to a weak promoter (a truncated
(-90 bp) CaMV35S promoter), can be used to identify tissue-specific
markers
in transgenic tobacco plants. The rationale was that the expression
of
gusA would be determined primarily by position effect. Quantitative
analysis revealed that, of 184 -90-gus transgenic plants, 73% exhibited
gusA gene
activation in leaf tissue, and the level of GUS enzyme activity
varied over a 300-fold range within the population. In comparison,
transformation
with a promoterless gusA gene resulted in GUS expression in 78%
of all plants analyzed (in leaf and/or root) and expression levels
were
three-fold
or more lower. Qualitative GUS analysis of single locus -90-gus
transformants revealed differential expression in diverse tissues.
The spatial pattern
of GUS activity was unique to individual transformants, was a reflection
of differential gusA gene transcription, and was stably transmissible
to progeny. Evidence for preferential expression in roots not only
of the -90-gus, but also the promoterless gusA gene is presented.
The value
of the -90 bp promoter-gusA sequence, which is termed an 'interposon',
as a tool both to identify native enhancer sequences in situ and
to investigate position effects in plants, is discussed.
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