Various Eyes Genetics BISC219 Wellesley CollegeBiology Department

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Laboratory Procedures

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]

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.
    : 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:

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.

Created by: Courtney Chin '07 & Christina L. Miller '08
Maintained by: Andrea Sequeira & Tucker Crum
Date created: July 13, 2005
Last modified: August 10, 2005
Expires: May 21, 2007