Molecular Genetics

BIOL 1540/2540
2016 Reading and Key Concepts List

Thursday 1/28/16 Course Overview

Tuesday 2/2/16 Yeast Cell Cycle screen

Primary Paper:
Genetic control of the cell-division cycle in yeast. I. Detection of mutants. Hartwell LH, Culotti J, Reid
B.
Proc Natl Acad Sci U S A. 1970 Jun;66(2):352-9. PMID: 5271168 [PubMed - indexed for MEDLINE]

Key Concepts:
How temperature-conditional mutations allow the study of lethal genes
Why studies in a single-celled fungus can be relevant to multi-cellular organisms including
humans
Advantages and disadvantages of doing genetics with haploid cells

Background Reading:
Macromolecule synthesis in temperature-sensitive mutants of yeast. Hartwell LH.
J Bacterial. 1967 May;93(5):1662-70.
PMID: 5337848 [PubMed - indexed for MEDLINE]

Pre-discussion Assignment: answer to posted question submitted on Canvas by 2:15 pm.

Thursday 2/4/16 C.elegans I

Primary Paper:
The genetics of Caenorhabditis elegans.
Brenner S.
Genetics. 1974 May;77(1):71-94.
PMID: 4366476 [PubMed - indexed for MEDLINE]

Key Concepts:
Considerations behind selection of a model organism to address a particular biological
question
Genetic advantages of a self-fertile organism
Patterns of inheritance for different types of mutations (autosomal versus sex-linked; recessive
versus dominant)
How to place recessive mutations into complementation groups.
How to construct a linkage map

Pre-discussion Assignment: answer to posted question submitted on Canvas by 2:15 pm.

Tuesday 2/9/16 C. elegans II

Primary Paper: (same as above):
The genetics of Caenorhabditis elegans.

1
Brenner S.
Genetics. 1974 May;77(1):71-94.
PMID: 4366476 [PubMed - indexed for MEDLINE]

Pre-discussion Assignment: In-class quiz.

Thursday 2/11/16 Arabidopsis flower development

Primary Paper:
Genetic interactions among floral homeotic genes of Arabidopsis.
Bowman JL, Smyth DR, Meyerowitz EM. Development. 1991 May;112(1):1-20.
PMID: 1685111

Key Concepts:
The utility of having multiple alleles with different strengths for a gene of interest
How double (or more) mutant analysis can be used to test hypotheses about gene interactions or
pathways
Use of transgene constructs to examine the effects of ectopic (abnormal in terms of level or
spatial/temporal patterns) gene expression

Pre-discussion Assignment: answer to posted question submitted on Canvas by 2:15 pm.

Tuesday 2/16/16 Arabidopsis ethylene signaling

Primary Paper:
Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana.
Hua J, Meyerowitz EM.
Cell. 1998 Jul 24;94(2):261-71.
PMID: 9695954 [PubMed - indexed for MEDLINE]

Key Concepts:
How one can analyze the function of a gene family using multiple loss-of-function mutants
Different ways that the function of a gene product can be altered to generate a dominany mutation
How to conduct a screen for intragenic suppressors of a dominant mutation
How careful design of mutant screens for opposite phenotypes can identify multiple factors in a
pathway of interes

Pre-discussion Assignment: answer to posted question submitted on Canvas by 2:15 pm.

Thursday 2/18/16 Yeast mating type switching

Primary Paper:
Epigenetic inheritance of transcriptional states in S. cerevisiae. Pillus L, Rine J.
Cell. 1989 Nov 17:59(4):637-47.
PMID: 2684414 [PubMed-indexed for MEDLINE]

Key Concepts:
Genetically identical cells can exist in phenotypically different states
Mitotically stable states can be generated without genetic changes
trans-acting factors are involved in mediating mitotically stable states

Pre-discussion Assignment: answer to posted question submitted on Canvas by 2:15 pm.

2
Tuesday 2/23/16 LONG WEEKEND

Thursday 2/25/16 MID-TERM EXAM I

Tuesday 3/1/16 Drosophila embryo lethal screen I

Primary Paper:
Mutations affecting the pattern of the larval cuticle in Drospholia melanogaster.
Nsslein-Volhard C, Wieschaus E., Kluding H Rouxs Arch Dev Biol. 1984 193:267-282

Key Concepts:
The patterns of inheritance for genes that are required during oogenesis (maternal effect lethals)
versus genes that are required after fertilization (zygotic lethals)
How balancer chromosomes aid in generating a homozygous mutation in non-self-fertile
organisms
Why balancer chromosomes are important for recovery and propagation of embryo- lethal
mutations
Why an inverted chromosomal segment is a key feature of a balancer chromosome
Criteria for determining whether a genetic screen is saturated

Background Reading:
Introduction to Genetic Analysis (Griffiths et al.)
This textbook provides a nice background on chromosomal aberrations that are used extensively in
Drosophila genetics. Pay careful attention the description of inversions and balancer chromosomes. There
is also a nice description in this textbook of some of the major paradigms of developmental biology that
came out of the work of Nsslein-Volhard and others. Additional reading:
Mutations affecting segment number and polarity in Drosophila. Nsslein-Volhard C, Wieschaus E.
Nature. 1980 Oct 30;287(5785):795-801.
PMID: 6776413 [PubMed - indexed for MEDLINE]

Pre-discussion Assignment: answer to posted question submitted on Canvas by 2:15 pm.

Thursday 3/3/16 Drosophila embryo lethal screen II

Primary Paper (same as above):
Mutations affecting the pattern of the larval cuticle in Drospholia melanogaster. Nsslein-Volhard C,
Wieschaus E., Kluding H
Rouxs Arch Dev Biol. 1984 193:267-282

Pre-discussion Assignment: In-class quiz.

Tuesday 3/8/16 Modifier screens

Primary Paper:
Genetic dissection of a neurodevelopmental pathway: Son of sevenless functions downstream of the
sevenless and EGF receptor tyrosine kinases.
Rogge RD, Karlovich CA, Banerjee U. Cell. 1991 Jan 11;64(1):39-48.
PMID: 1846090 [PubMed - indexed for MEDLINE]

3
Secondary Paper:
Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the
sevenless protein tyrosine kinase.
Simon MA, Bowtell DD, Dodson GS, Laverty TR, Rubin GM. Cell. 1991 Nov 15;67(4):701-16.
PMID: 1934068 [PubMed - indexed for MEDLINE]

Key Concepts:
How modifier screens allow recovery of mutations in genes that are lethal or redundant
Why dominant mutations might be misleading about the normal function of a gene, and how this
possibility could be addressed experimentally
The logic, design, and utility of mosaic analysis
Why a temperature-sensitive mutant grown at the permissive temperature is a good starting strain
for a modifier screen

Pre-discussion Assignment: answer to posted question submitted on Canvas by 2:15 pm.

Thursday 3/10/16 MicroRNAs

Primary Paper:
The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.
Lee RC, Feinbaum RL, Ambros V. Cell. 1993 Dec 3;75(5):843-54.
PMID: 8252621 [PubMed - indexed for MEDLINE]

Key Concepts:
The multiple modes of gene regulation that can be mediated by an argonaute/small RNA complex
Why only a single allele of lin-4 was isolated in the exhaustive initial screens for heterochronic
mutations
How a non-complementation screen can be used to identify additional recessive alleles in a gene
of interest
How transgenes can be used to validate candidate miRNA targets

Historically interesting and insightful commentary: The evolution of our thinking about microRNAs.
Ambros V.
Nat Med. 2008 Oct;14(10):1036-40. No abstract available. PMID: 18841144 [PubMed - indexed for
MEDLINE]

Pre-discussion Assignment: submit Haiku on Canvas by 2:15 pm.

See wikipedia for some examples of translations from Bash (3 lines, 5 syllables in first line, 7 syllables in
second line, 5 syllables in third line).
http://en.wikipedia.org/wiki/Haiku
We will read a couple of haiku before class that capture the essence of the focus papers.

Tuesday 3/15/16 RNA interference

Primary Paper:
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Nature. 1998 Feb 19;391(6669):806-11.
PMID: 9486653 [PubMed - indexed for MEDLINE]

Key Concepts:
Why RNAi is both potent and specific
How RNAi can cause unintended down-regulation of off-target genes

4
 That RNAi may be systemically spread throughout an organism or even (in C. elegans) be
inherited
The threshold model for how sense-suppresssion/co-suppression in plants is mediated by RNA-
dependent RNA polymerases

Secondary Paper:
A species of small antisense RNA in posttranscriptional gene silencing in plants. Hamilton AJ, Baulcombe
DC.
Science. 1999 Oct 29;286(5441):950-2.
PMID: 10542148 [PubMed - indexed for MEDLINE]

Pre-discussion Assignment: answer to posted question submitted on Canvas by 2:15 pm.

Tuesday 3/17/16 Genome-wide RNAi

Primary Paper:
Functional genomic analysis of RNA interference in C. elegans.
Kim JK, Gabel HW, Kamath RS, Tewari M, Pasquinelli A, Rual JF, Kennedy S, Dybbs M, Bertin N, Kaplan
JM, Vidal M, Ruvkun G.
Science. 2005 May 20;308(5725):1164-7. Epub 2005 Mar 24.PMID: 15790806

Key Concepts:
How microarray and Next Generation Sequencing strategies allow genome-wide surveys
Strengths and weaknesses of genome-wide deletion mutant, insertion mutant, or RNAi collections
versus traditional forward genetic screens
Why different screens for RNAi pathway components do not always identify the same factors
Why RNAi analysis of lethal genes in C. elegans or cultured cells can yield mutants with indirect
effects on the pathway of interest

Pre-discussion Assignment: answer to posted question submitted on Canvas by 2:15 pm.

Tuesday 3/22/16 Zebrafish w/Robert Creton

Thursday 3/24/16 MID-TERM EXAM II

Tuesday 3/29/16 SPRING BREAK

Thursday 3/31/16 SPRING BREAK

Tuesday 4/5/16 Huntington Disease I

Primary Paper:
A polymorphic DNA marker genetically linked to Huntington's disease.
Gusella JF, Wexler NS, Conneally PM, Naylor SL, Anderson MA, Tanzi RE, Watkins PC, Ottina K, Wallace
MR, Sakaguchi AY, et al.
Nature. 1983 Nov 17-23;306(5940):234-8.
PMID: 6316146

5
Background Reading:
An Introduction to Human Molecular Genetics: Mechanisms of Inherited Disease, Second Edition, by Jack
J. Pasternak.
Available online at: http://site.ebrary.com/lib/brown/docDetail.action?docID=10113938 or alternatively at
http://josiah.brown.edu/record=b5356977.
To access these sites off campus, you need to use one of the off-site campus programs such as EZProxy.
Chapter 3 and Chapter 6 in complement to the lecture, these recommended chapters will be helpful
background for building a foundation in human genetics.

Key Concepts:
How does human genetics differ from genetics in model organisms?
What do we mean by the genetic architecture of a human phenotype?
When we try to map a human phenotype we need to form a hypothesis about the genetic
architecture of the phenotype. What data should be incorporated into this hypothesis?
What is the nature and variety of genetic variation in the human genome?
What constitutes statistical proof for association of a genetic variant with phenotype in a human
linkage study?

Pre-discussion Assignment: answer to posted question submitted on Canvas by 2:15 pm.

Thursday 4/7/16 Huntingtons Disease II

Primary Paper:
A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease
chromosomes. The Huntington's Disease Collaborative Research Group.
[MacDonald and many others] Cell. 1993 Mar 26;72(6):971-83.
PMID: 8458085

Additional Reading:
Huntington's disease: seeing the pathogenic process through a genetic lens. Gusella JF, MacDonald ME.
Trends Biochem Sci. 2006 Sep;31(9):533-40. PMID: 16829072

Key Concepts:
What tools do we need in the toolbox to go from linkage to mutation that is, why did it take 10
years to find the gene?
De novo vs inherited mutation ie the genome is both dynamic and inherited!
Trinucleotide repeat expansion as a molecular basis for anticipation
What are the ethical considerations in human genetic testing

Pre-discussion Assignment: answer to posted question submitted on Canvas by 2:15 pm.

Tuesday 4/12/16 Targeted gene disruption in mouse

Primary Paper:
Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting
mutations to non-selectable genes.
Mansour SL, Thomas KR, Capecchi MR. Nature 1988 Nov 24;336:348-352.

Key Concepts:
How site-specific recombination systems work
How to alter both alleles of a gene in a diploid ES cell line

6
 A range of genetic engineering techniques in mouse
Potential pitfalls associated with tissue-specific gene replacements
How to use genetic engineering in mouse to ask questions about development

Pre-discussion Assignment: In-class quiz.

Thursday 4/14/16 Conditional gene disruption in mouse

Primary Paper:
Reversal of neurological defects in a mouse model of Rett syndrome.
Guy J, Gan J, Selfridge J, Cobb S, Bird A
Science 2007 Feb 23;315(5815):1143-7
PMID: 17289941

Key Concepts:
Transgenic mouse genetics to combine the conditional construct with a tissue-specific
recombinase
Inducible mutations
Selective use of these techniques to address specific scientific questions or model disease

Pre-discussion Assignment: answer to posted question submitted on Canvas by 2:15 pm.

Tuesday 4/19/16 Intellectual Disability genetics

Primary Paper:
A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.
Tarpey PS, Smith R, Pleasance E, Whibley A, Edkins S, Hardy C, O'Meara S, Latimer C, Dicks E,
Menzies A, Stephens P, Blow M, Greenman C, Xue Y, Tyler-Smith C, Thompson D, Gray K, Andrews
J, Barthorpe S, Buck G, Cole J, Dunmore R, Jones D, Maddison M, Mironenko T, Turner R, Turrell K,
Varian J, West S, Widaa S, Wray P, Teague J, Butler A, Jenkinson A, Jia M, Richardson D, Shepherd
R, Wooster R, Tejada Ml, Martinez F, Carvill G, Goliath R, de Brouwer AP, van Bokhoven H, Van
Esch H, Chelly J, Raynaud M, Ropers HH, Abidi FE, Srivastava AK, Cox J, Luo Y, Mallya U, Moon J,
Parnau J, Mohammed S, Tolmie JL, Shoubridge C, Corbett M, Gardner A, Haan E, Rujirabanjerd S,
Shaw M, Vandeleur L, Fullston T, Easton OF, Boyle J, Partington M, Hackett A, Field M, Skinner C,
Stevenson RE, Bobrow M, Turner G, Schwartz CE, Gecz J, Raymond FL, Futreal PA, Stratton MR.
Nat Genet. 2009 May;41(5):535-43. Epub 2009 Apr 19.
PMID: 19377476

Additional Reading:
The genetic landscape of intellectual disability arising from chromosome X. Gecz J, Shoubridge C,
Corbett M.
Trends Genet. 2009 Jul;25(7):308-16. PMID: 19556021

Key Concepts:
What is the definition of genetic heterogeneity?
What is the definition of syndromic versus non-syndromic intellectual disability? What is the
significance of these phenotypic features to mutation discovery?
How does the concept of variable expressivity relate to phenotype of ID? How is this related to the
concept of incomplete penetrance.
Discuss the relevance of rare genetic variation versus common genetic variation in the context
of intellectual disability.
What constitutes statistical proof for association of a genetic variant with phenotype in

7
 sequencing studies

Pre-discussion Assignment: In-class quiz.

Thursday 4/21/16 Induced pluripotent stem cells and genome-editing

Primary Paper:
Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point
mutations
Soldner F, Laganire J, Cheng AW, Hockemeyer D, Gao Q, Alagappan R, Khurana V, Golbe LI, Myers
RH, Lindquist S, Zhang L, Guschin D, Fong LK, Vu BJ, Meng X, Urnov FD, Rebar EJ, Gregory PD, Zhang
HS, Jaenisch R
Cell 2011 Jul 22;146(2):318-31; Erratum in Cell 2011 Aug 19;146(4):659
PMID: 21757228

Background Reading:
ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering
Gaj T, Gersbach CA, Barbas CF 3rd
Trends Biotechnol 2013 Jul;31(7):397-405
PMID: 23664777

Induced pluripotency: history, mechanisms, and applications

Stadtfeld M, Hochedlinger K
Genes Dev 2010 Oct 15;24(20):2239-63
PMID: 20952534

Key Concepts:
The importance of Nuclear Reprogramming technology to the study of human disease
Molecular mechanisms underlying the generation of induced pluripotent stem cells
The molecular basis for the different Genome editing methods (ZFN, TALENs and CRISPR/CAS
The relevance to using isogenic controls to study disease

Pre-discussion Assignment: Paper Summary submitted on Canvas by 2:15 pm.

Tuesday 4/26/16 Student Presentations I

Thursday 4/28/16 Student Presentations II