Genetics Commons^™

Physical Interaction Between Vivid And White Collar Complex Regulates Photoadaptation In Neurospora, Chen-Hui H. Chen, Bradley S. Demay, Amy S. Gladfelter, Jay Dunlap, Jennifer J. Loros

Dartmouth Scholarship

Photoadaptation, the ability to attenuate a light response on prolonged light exposure while remaining sensitive to escalating changes in light intensity, is essential for organisms to decipher time information appropriately, yet the underlying molecular mechanisms are poorly understood. In Neurospora crassa, VIVID (VVD), a small LOV domain containing blue-light photoreceptor protein, affects photoadaptation for most if not all light-responsive genes. We report that there is a physical interaction between VVD and the white collar complex (WCC), the primary blue-light photoreceptor and the transcription factor complex that initiates light-regulated transcriptional responses in Neurospora. Using two previously characterized VVD mutants, we show …

Quantifying And Resolving Multiple Vector Transformants In S. Cerevisiae Plasmid Libraries, Thomas C. Scanlon, Elizabeth C. Gray, Karl E. Griswold

Dartmouth Scholarship

In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the …

Constructing And Using Case Studies In Genetics To Engage Students In Active Learning, Sue Styer

Faculty Publications & Research

One of the national goals in science education is to teach science in a way that mirrors the process of science as inquiry, described by the National Science Education Standards (NSES) Science Teaching Standard B and Content Standard A (NRC, 1996). Inquiry-based learning, including the use of case studies, is one of several types of active learning that allows students to experience critical thinking skills inherent in the science process (Handelsman et al., 2007). Using case studies also develops skills in group learning and personalizes and humanizes science, making it more relevant to students (National Center for Case Study Teaching …

Efficient Gene Replacements In Toxoplasma Gondii Strains Deficient For Nonhomologous End Joining, Barbara A. Fox, Jessica G. Ristuccia, Jason P. Gigley, David J. Bzik

Dartmouth Scholarship

A high frequency of nonhomologous recombination has hampered gene targeting approaches in the model apicomplexan parasite Toxoplasma gondii. To address whether the nonhomologous end-joining (NHEJ) DNA repair pathway could be disrupted in this obligate intracellular parasite, putative KU proteins were identified and a predicted KU80 gene was deleted. The efficiency of gene targeting via double-crossover homologous recombination at several genetic loci was found to be greater than 97% of the total transformants in KU80 knockouts. Gene replacement efficiency was markedly increased (300- to 400-fold) in KU80 knockouts compared to wild-type strains. Target DNA flanks of only approximately 500 bp were …

Evolution Acts On Enhancer Organization To Fine-Tune Gradient Threshold Readouts, Justin Crocker, Yoichiro Tamori, Albert Erives

Dartmouth Scholarship

The elucidation of principles governing evolution of gene regulatory sequence is critical to the study of metazoan diversification. We are therefore exploring the structure and organizational constraints of regulatory sequences by studying functionally equivalent cis-regulatory modules (CRMs) that have been evolving in parallel across several loci. Such an independent dataset allows a multi-locus study that is not hampered by nonfunctional or constrained homology. The neurogenic ectoderm enhancers (NEEs) of Drosophila melanogaster are one such class of coordinately regulated CRMs. The NEEs share a common organization of binding sites and as a set would be useful to study the relationship …

Ovarian Development In Mice Requires The Gata4-Fog2 Transcription Complex, Nikolay L. Manuylov, Fatima O. Smagulova, Lyndsay Leach, Sergei G. Tevosian

Dartmouth Scholarship

We have demonstrated previously that mammalian sexual differentiation requires both the GATA4 and FOG2 transcriptional regulators to assemble the functioning testis. Here we have determined that the sexual development of female mice is profoundly affected by the loss of GATA4-FOG2 interaction. We have also identified the Dkk1 gene, which encodes a secreted inhibitor of canonical beta-catenin signaling, as a target of GATA4-FOG2 repression in the developing ovary. The tissue-specific ablation of the beta-catenin gene in the gonads disrupts female development. In Gata4(ki/ki); Dkk1(-/-) or Fog2(-/-); Dkk1(-/-) embryos, the normal ovarian gene expression pattern is partially restored. Control of ovarian development …

Systems Approach Identifies An Organic Nitrogen-Responsive Gene Network That Is Regulated By The Master Clock Control Gene Cca1, Rodrigo A. Gutierrez, Trevor L. Stokes, Karen Thum, Xiaodong Xu, Mariana Obertello, Manpreet S. Katari, Milos Tanurdzic, Alexis Dean, Damion C. Nero, C Robertson Mcclung, Gloria M. Coruzzi

Dartmouth Scholarship

Understanding how nutrients affect gene expression will help us to understand the mechanisms controlling plant growth and development as a function of nutrient availability. Nitrate has been shown to serve as a signal for the control of gene expression in Arabidopsis. There is also evidence, on a gene-by-gene basis, that downstream products of nitrogen (N) assimilation such as glutamate (Glu) or glutamine (Gln) might serve as signals of organic N status that in turn regulate gene expression. To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of …

Variation In Molybdenum Content Across Broadly Distributed Populations Of Arabidopsis Thaliana Is Controlled By A Mitochondrial Molybdenum Transporter (Mot1), Ivan Baxter, Balasubramaniam Muthukumar, Hyeong Cheol Park, Peter Buchner, Brett Lahner, John Danku, Keyan Zhao, Joohyun Lee, Malcolm J. Hawkesford, Mary Lou Guerinot, David E. Salt

Dartmouth Scholarship

Molybdenum (Mo) is an essential micronutrient for plants, serving as a cofactor for enzymes involved in nitrate assimilation, sulfite detoxification, abscisic acid biosynthesis, and purine degradation. Here we show that natural variation in shoot Mo content across 92 Arabidopsis thaliana accessions is controlled by variation in a mitochondrially localized transporter (Molybdenum Transporter 1 - MOT1) that belongs to the sulfate transporter superfamily. A deletion in the MOT1 promoter is strongly associated with low shoot Mo, occurring in seven of the accessions with the lowest shoot content of Mo. Consistent with the low Mo phenotype, MOT1 expression in low Mo accessions …

Fully Codon-Optimized Luciferase Uncovers Novel Temperature Characteristics Of The Neurospora Clock, Van D. Gooch, Arun Mehra, Luis F. Larrondo, Julie Fox, Melissa Touroutoutoudis, Jennifer J. Loros, Jay C. Dunlap

Dartmouth Scholarship

We report the complete reconstruction of the firefly luciferase gene, fully codon optimized for expression in Neurospora crassa. This reporter enhances light output by approximately 4 log orders over that with previously available versions, now producing light that is visible to the naked eye and sufficient for monitoring the activities of many poorly expressed genes. Time lapse photography of strains growing in race tubes, in which the frq or eas/ccg-2 promoter is used to drive luciferase, shows the highest levels of luciferase activity near the growth front and newly formed conidial bands. Further, we have established a sorbose medium colony …

Single-Stranded Dna-Binding Proteins Regulate The Abundance Of Lim Domain And Lim Domain-Binding Proteins, Zhixiong Xu, Xianzhang Meng, Ying Cai, Hong Liang, Lalitha Nagarajan, Stephen J Brandt

Faculty, Staff and Student Publications

The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In …

Similar Levels Of X-Linked And Autosomal Nucleotide Variation In African And Non-African Populations Of Drosophila Melanogaster, Nadia D. Singh, J. Michael Macpherson, Jeffrey D. Jensen, Dmitri A. Petrov

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Background: Levels of molecular diversity in Drosophila have repeatedly been shown to be higher in ancestral, African populations than in derived, non-African populations. This pattern holds for both coding and noncoding regions for a variety of molecular markers including single nucleotide polymorphisms and microsatellites. Comparisons of X-linked and autosomal diversity have yielded results largely dependent on population of origin.

Results: In an attempt to further elucidate patterns of sequence diversity in Drosophila melanogaster, we studied nucleotide variation at putatively nonfunctional X-linked and autosomal loci in sub-Saharan African and North American strains of D. melanogaster. We combine our experimental …

A Subset Of Arabidopsis Ap2 Transcription Factors Mediates Cytokinin Responses In Concert With A Two-Component Pathway, Aaron M. Rashotte, Michael G. Mason, Claire E. Hutchison, Fernando J. Ferreira, G. Eric Schaller, Joseph J. Kieber

Dartmouth Scholarship

The plant hormone cytokinin regulates numerous growth and developmental processes. A signal transduction pathway for cytokinin has been elucidated that is similar to bacterial two-component phosphorelays. In Arabidopsis, this pathway is comprised of receptors that are similar to sensor histidine kinases, histidine-containing phosphotransfer proteins, and response regulators (ARRs). There are two classes of response regulators, the type-A ARRs, which act as negative regulators of cytokinin responses, and the type-B ARRs, which are transcription factors that play a positive role in mediating cytokinin-regulated gene expression. Here we show that several closely related members of the Arabidopsis AP2 gene family of …

Saccharomyces Cerevisiae-Based Molecular Tool Kit For Manipulation Of Genes From Gram-Negative Bacteria, Robert M. Q. Shanks, Nicky C. Caiazza, Shannon M. Hinsa, Christine M. Toutain, George A. O'Toole

Dartmouth Scholarship

A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface …

Bounded Search For De Novo Identification Of Degenerate Cis-Regulatory Elements, Jonathan M. Carlson, Arijit Chakravarty, Radhika S. Khetani, Robert H. Gross

Dartmouth Scholarship

The identification of statistically overrepresented sequences in the upstream regions of coregulated genes should theoretically permit the identification of potential cis-regulatory elements. However, in practice many cis-regulatory elements are highly degenerate, precluding the use of an exhaustive word-counting strategy for their identification. While numerous methods exist for inferring base distributions using a position weight matrix, recent studies suggest that the independence assumptions inherent in the model, as well as the inability to reach a global optimum, limit this approach.

A Role For Cetp Taqib Polymorphism In Determining Susceptibility To Atrial Fibrillation: A Nested Case Control Study, Folkert W. Asselbergs, Jason H. Moore, Maarten P. Van Den Berg, Eric B. Rimm

Dartmouth Scholarship

Studies investigating the genetic and environmental characteristics of atrial fibrillation (AF) may provide new insights in the complex development of AF. We aimed to investigate the association between several environmental factors and loci of candidate genes, which might be related to the presence of AF. A nested case-control study within the PREVEND cohort was conducted. Standard 12 lead electrocardiograms were recorded and AF was defined according to Minnesota codes. For every case, an age and gender matched control was selected from the same population (n = 194). In addition to logistic regression analyses, the multifactor-dimensionality reduction (MDR) method and interaction …

Principal Component Analysis For Predicting Transcription-Factor Binding Motifs From Array-Derived Data, Yunlong Liu, Matthew P Vincenti, Hiroki Yokota

Dartmouth Scholarship

The responses to interleukin 1 (IL-1) in human chondrocytes constitute a complex regulatory mechanism, where multiple transcription factors interact combinatorially to transcription-factor binding motifs (TFBMs). In order to select a critical set of TFBMs from genomic DNA information and an array-derived data, an efficient algorithm to solve a combinatorial optimization problem is required. Although computational approaches based on evolutionary algorithms are commonly employed, an analytical algorithm would be useful to predict TFBMs at nearly no computational cost and evaluate varying modelling conditions. Singular value decomposition (SVD) is a powerful method to derive primary components of a given matrix. Applying SVD …

Estimation Of The Incidence Of A Rare Genetic Disease Through A Two-Tier Mutation Survey, R Chakraborty, M R Srinivasan, S Raskin

Faculty, Staff and Student Publications

Recent attempts to detect mutations involving single base changes or small deletions that are specific to genetic diseases provide an opportunity to develop a two-tier mutation-screening program through which incidence of rare genetic disorders and gene carriers may be precisely estimated. A two-tier survey consists of mutation screening in a sample of patients with specific genetic disorders and in a second sample of newborns from the same population in which mutation frequency is evaluated. We provide the statistical basis for evaluating the incidence of affected and gene carriers in such two-tier mutation-screening surveys, from which the precision of the estimates …

Population Amalgamation And Genetic Variation: Observations On Artificially Agglomerated Tribal Populations Of Central And South America, R Chakraborty, P E Smouse, J V Neel

Faculty, Staff and Student Publications

The interpretation of data on genetic variation with regard to the relative roles of different evolutionary factors that produce and maintain genetic variation depends critically on our assumptions concerning effective population size and the level of migration between neighboring populations. In humans, recent population growth and movements of specific ethnic groups across wide geographic areas mean that any theory based on assumptions of constant population size and absence of substructure is generally untenable. We examine the effects of population subdivision on the pattern of protein genetic variation in a total sample drawn from an artificial agglomerate of 12 tribal populations …

International Registry Of Poultry Genetic Stocks, Ralph G. Somes Jr.

Storrs Agricultural Experiment Station

No abstract provided.

Parentage Analysis With Genetic Markers In Natural Populations I The Expected Proportion Of Offspring With Unambiguous Paternity, R Chakraborty, T R Meagher, P E Smouse

Faculty, Staff and Student Publications

Recent studies indicate that polymorphic genetic markers are potentially helpful in resolving genealogical relationships among individuals in a natural population. Genetic data provide opportunities for paternity exclusion when genotypic incompatibilities are observed among individuals, and the present investigation examines the resolving power of genetic markers in unambiguous positive determination of paternity. Under the assumption that the mother for each offspring in a population is unambiguously known, an analytical expression for the fraction of males excluded from paternity is derived for the case where males and females may be derived from two different gene pools. This theoretical formulation can also be …

Cole1 Copy Number Mutants., Londa Schmidt, Joseph Inselburg

Dartmouth Scholarship

A deletion mutant of the colicin E1-derived plasmid, pDMS6642, exhibited an approximately fourfold increase in copy number. We subsequently isolated hydroxylamine-induced mutants of that plasmid that had a further increase in copy number. Analysis of them suggests that the increased copy number of pDMS6642 is associated with transcriptional readthrough from a Tn3 transposon into the region of ColE1 containing information that influences plasmid replication. The hydroxylamine mutation in one copy number mutant appeared to increase the plasmid copy number by stimulating readthrough transcription from the Tn3 transposon into the ColE1 replication control region, whereas the other hydroxylamine mutation acts by …

Genetically Controlled Variation Of “Acid” Beta-Galactosidase Detected In Rattus Norvegicus By Isoelectric Focusing, T C Douglas, K A Kimmel, P E Dawson

Faculty, Staff and Student Publications

Two genetically variant forms of rat "acid" beta-galactosidase were found to differ in isoelectric point and pH dependence, but not in thermostability or sensitivity to inhibition by p-mercuribenzoate (PMB). The results of two backcrosses and an intercross indicated that the isoelectric focusing phenotypes are controlled by two codominant alleles at a single autosomal locus, for which we propose the name Glb-1. No significant linkage between Glb-1 and albino (LG I), brown (LG II), or hooded (LG VI) was observed. Strain-specific differences in total levels of kidney beta-galactosidase were detected, but it is not yet known whether the variation is controlled …

Polymorphisms, Linkage And Mapping Of Four Enzyme Loci In The Fish Genus Xiphophorus (Poeciliidae), D C Morizot, M J Siciliano

Faculty, Staff and Student Publications

Electrophoretic variants at four additional enzyme loci--two esterases (Est-2, Est-3), retinal lactate dehydrogenase (LDH-1) and mannose phosphate isomerase (MPI)--among three species and four subspecies of fish of the genus Xiphophorus were observed. Electrophoretic patterns in F1 hybrid heterozygotes confirmed the monomeric structures of MPI and the esterase and the tetrametric structure of LDH in these fishes. Variant alleles of all four loci displayed normal Mendelian segregation in backcross and F2 hybrids. Recombination data from backcross hybrids mapped with Haldane's mapping function indicate the four loci to be linked as Est-2--0.43--Est3--0.26--LDH-1--0.19--MPI. Significant interference was detected and apparently concentrated in the Est-3 …

Three Linked Enzyme Loci In Fishes: Implications In The Evolution Of Vertebrate Chromosomes, D C Morizot, D A Wright, M J Siciliano

Faculty, Staff and Student Publications

A three-point linkage group comprised of loci coding for adenosine deaminase (ADA), glucose-6-phosphate dehydrogenase (G6PDH), and 6-phospho-gluconate dehydrogenase (6PGD) is described in fish of the genus Xiphophorus (Poeciliidae). The alleles at loci in this group were shown to assort independently from the alleles at three other loci--isocitrate dehydrogenase 1 and 2, and glyceraldehyde-3-phosphate dehydrogenase 1. Alleles at the latter three loci also assort independently from each other. Data were obtained by observing the segregation of electrophoretically variant alleles in reciprocal backcross hybrids derived from crosses between either X. helleri guentheri or X. h. strigatus and X. maculatus. The linkage component …