Medicine and Health Sciences Commons^™

An Expanded Toolkit For Gene Tagging Based On Mimic And Scarless Crispr Tagging In, David Li-Kroeger, Oguz Kanca, Pei-Tseng Lee, Sierra Cowan, Michael T Lee, Manish Jaiswal, Jose Luis Salazar, Yuchun He, Zhongyuan Zuo, Hugo J Bellen

Faculty Publications

We generated two new genetic tools to efficiently tag genes in Drosophila. The first, Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on Cre recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced …

Identification Of A Novel Gene On 10q22.1 Causing Autosomal Dominant Retinitis Pigmentosa (Adrp)., Stephen P Daiger, Lori S Sullivan, Sara J Bowne, Daniel C Koboldt, Susan H Blanton, Dianna K Wheaton, Cheryl E Avery, Elizabeth D Cadena, Robert K Koenekoop, Robert S Fulton, Richard K Wilson, George M Weinstock, Richard A Lewis, David G Birch

Faculty Publications

Whole-genome linkage mapping identified a region on chromosome 10q21.3-q22.1 with a maximum LOD score of 3.0 at 0 % recombination in a six-generation family with autosomal dominant retinitis pigmentosa (adRP). All known adRP genes and X-linked RP genes were excluded in the family by a combination of methods. Whole-exome next-generation sequencing revealed a missense mutation in hexokinase 1, HK1 c.2539G > A, p.Glu847Lys, tracking with disease in all affected family members. One severely-affected male is homozygous for this region by linkage analysis and has two copies of the mutation. No other potential mutations were detected in the linkage region nor were …

Large-Scale Identification Of Chemically Induced Mutations In Drosophila Melanogaster., Nele A Haelterman, Lichun Jiang, Yumei Li, Vafa Bayat, Hector Sandoval, Berrak Ugur, Kai Li Tan, Ke Zhang, Danqing Bei, Bo Xiong, Wu-Lin Charng, Theodore Busby, Adeel Jawaid, Gabriela David, Manish Jaiswal, Koen J T Venken, Shinya Yamamoto, Rui Chen, Hugo J Bellen

Faculty Publications

Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of …

Highly Variable Recessive Lethal Or Nearly Lethal Mutation Rates During Germ-Line Development Of Male Drosophila Melanogaster., Jian-Jun Gao, Xue-Rong Pan, Jing Hu, Li Ma, Jian-Min Wu, Ye-Lin Shao, Sara A Barton, Ronny C Woodruff, Ya-Ping Zhang, Yun-Xin Fu

Journal Articles

Each cell of higher organism adults is derived from a fertilized egg through a series of divisions, during which mutations can occur. Both the rate and timing of mutations can have profound impacts on both the individual and the population, because mutations that occur at early cell divisions will affect more tissues and are more likely to be transferred to the next generation. Using large-scale multigeneration screening experiments for recessive lethal or nearly lethal mutations of Drosophila melanogaster and recently developed statistical analysis, we show for male D. melanogaster that (i) mutation rates (for recessive lethal or nearly lethal) are …

Enhanced Estrogen-Induced Proliferation In Obese Rat Endometrium, Qian Zhang, Qi Shen, Joseph Celestino, Michael R Milam, Shannon N Westin, Robin A Lacour, Larissa A Meyer, Gregory L Shipley, Peter J A Davies, Lei Deng, Adrienne S Mccampbell, Russell R Broaddus, Karen H Lu

Journal Articles

OBJECTIVE: We tested the hypothesis that the proliferative estrogen effect on the endometrium is enhanced in obese vs lean animals.

STUDY DESIGN: Using Zucker fa/fa obese rats and lean control, we examined endometrial cell proliferation and the expression patterns of certain estrogen-regulated proproliferative and antiproliferative genes after short-term treatment with estradiol.

RESULTS: No significant morphologic/histologic difference was seen between the obese rats and the lean rats. Estrogen-induced proproliferative genes cyclin A and c-Myc messenger RNA expression were significantly higher in the endometrium of obese rats compared with those of the lean control. Expression of the antiproliferative gene p27Kip1 was suppressed …

Regulation Of Survivin Gene Expression In The Human Endometrium And Endometrial Cancer, Nancy H. Nabilsi

Dissertations & Theses (Open Access)

In the United States, endometrial cancer is the leading cancer of the female reproductive tract. There are 40,100 new cases and 7,470 deaths from endometrial cancer estimated for 2008 (47). The average five year survival rate for endometrial cancer is 84% however, this figure is substantially lower in patients diagnosed with late stage, advanced disease and much higher for patients diagnosed in early stage disease (47). Endometrial cancer (EC) has been associated with several risk factors including obesity, diabetes, hypertension, previously documented occurrence of hereditary non-polyposis colorectal cancer (HNPCC), and heightened exposure to estrogen (25). As of yet, there has …

Identification And Phenotypic Characterization Of A Second Collagen Adhesin, Scm, And Genome-Based Identification And Analysis Of 13 Other Predicted Mscramms, Including Four Distinct Pilus Loci, In Enterococcus Faecium, Jouko Sillanpää, Sreedhar R Nallapareddy, Vittal P Prakash, Xiang Qin, Magnus Höök, George M Weinstock, Barbara E Murray

Journal Articles

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 …

A Functional Collagen Adhesin Gene, Acm, In Clinical Isolates Of Enterococcus Faecium Correlates With The Recent Success Of This Emerging Nosocomial Pathogen, Sreedhar R Nallapareddy, Kavindra V Singh, Pablo C Okhuysen, Barbara E Murray

Journal Articles

Enterococcus faecium recently evolved from a generally avirulent commensal into a multidrug-resistant health care-associated pathogen causing difficult-to-treat infections, but little is known about the factors responsible for this change. We previously showed that some E. faecium strains express a cell wall-anchored collagen adhesin, Acm. Here we analyzed 90 E. faecium isolates (99% acm(+)) and found that the Acm protein was detected predominantly in clinically derived isolates, while the acm gene was present as a transposon-interrupted pseudogene in 12 of 47 isolates of nonclinical origin. A highly significant association between clinical (versus fecal or food) origin and collagen adherence (P

Genes In Glucose Metabolism And Association With Spina Bifida, Christina M Davidson, Hope Northrup, Terri M King, Jack M Fletcher, Irene Townsend, Gayle H Tyerman, Kit Sing Au

Journal Articles

The authors test single nucleotide polymorphisms (SNPs) in coding sequences of 12 candidate genes involved in glucose metabolism and obesity for associations with spina bifida. Genotyping was performed on 507 children with spina bifida and their parents plus anonymous control DNAs from Hispanic and Caucasian individuals. The transmission disequilibrium test was performed to test for genetic associations between transmission of alleles and spina bifida in the offspring (P < .05). A statistically significant association between Lys481 of HK1 (G allele), Arg109Lys of LEPR (G allele), and Pro196 of GLUT1 (A allele) was found ( P = .019, .039, and .040, respectively). Three SNPs on 3 genes involved with glucose metabolism and obesity may be associated with increased susceptibility to spina bifida.

The Role Of Sse1 In The De Novo Formation And Variant Determination Of The [Psi+] Prion, Qing Fan, Kyung-Won Park, Zhiqiang Du, Kevin A Morano, Liming Li

Journal Articles

Yeast prions are a group of non-Mendelian genetic elements transmitted as altered and self-propagating conformations. Extensive studies in the last decade have provided valuable information on the mechanisms responsible for yeast prion propagation. How yeast prions are formed de novo and what cellular factors are required for determining prion "strains" or variants--a single polypeptide capable of existing in multiple conformations to result in distinct heritable phenotypes--continue to defy our understanding. We report here that Sse1, the yeast ortholog of the mammalian heat-shock protein 110 (Hsp110) and a nucleotide exchange factor for Hsp70 proteins, plays an important role in regulating [PSI+] …

Inducible Caspase 9 Suicide Gene To Improve The Safety Of Allodepleted T Cells After Haploidentical Stem Cell Transplantation., Siok-Keen Tey, Gianpietro Dotti, Cliona M. Rooney, Helen E. Heslop, Malcolm K. Brenner

Faculty Publications

Addback of donor T cells following T cell-depleted stem cell transplantation (SCT) can accelerate immune reconstitution and be effective against relapsed malignancy. After haploidentical SCT, a high risk of graft-versus-host disease (GVHD) essentially precludes this option, unless the T cells are first depleted of alloreactive precursor cells. Even then, the risks of severe GVHD remain significant. To increase the safety of the approach and thereby permit administration of larger T cell doses, we used a suicide gene, inducible caspase 9 (iCasp9), to transduce allodepleted T cells, permitting their destruction should administration have adverse effects. We made a retroviral vector encoding …

Transcriptional Regulation Of The Borrelia Burgdorferi Antigenically Variable Vlse Surface Protein, Tomasz Bykowski, Kelly Babb, Kate Von Lackum, Sean P Riley, Steven J Norris, Brian Stevenson

Journal Articles

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. …

Comparison Of Og1rf And An Isogenic Fsrb Deletion Mutant By Transcriptional Analysis: The Fsr System Of Enterococcus Faecalis Is More Than The Activator Of Gelatinase And Serine Protease, Agathe Bourgogne, Susan G Hilsenbeck, Gary M Dunny, Barbara E Murray

Journal Articles

The FsrABC system of Enterococcus faecalis controls the expression of gelatinase and a serine protease via a quorum-sensing mechanism, and recent studies suggest that the Fsr system may also regulate other genes important for virulence. To investigate the possibility that Fsr influences the expression of additional genes, we used transcriptional profiling, with microarrays based on the E. faecalis strain V583 sequence, to compare the E. faecalis strain OG1RF with its isogenic mutant, TX5266, an fsrB deletion mutant. We found that the presence of an intact fsrB influences expression of numerous genes throughout the growth phases tested, namely, late log to …

Abnormal Expression Of Rest/Nrsf And Myc In Neural Stem/Progenitor Cells Causes Cerebellar Tumors By Blocking Neuronal Differentiation., Xiaohua Su, Vidya Gopalakrishnan, Duncan Stearns, Kenneth Aldape, Fredrick F Lang, Gregory Fuller, Evan Snyder, Charles G Eberhart, Sadhan Majumder

Journal Articles

Medulloblastoma, one of the most malignant brain tumors in children, is thought to arise from undifferentiated neural stem/progenitor cells (NSCs) present in the external granule layer of the cerebellum. However, the mechanism of tumorigenesis remains unknown for the majority of medulloblastomas. In this study, we found that many human medulloblastomas express significantly elevated levels of both myc oncogenes, regulators of neural progenitor proliferation, and REST/NRSF, a transcriptional repressor of neuronal differentiation genes. Previous studies have shown that neither c-Myc nor REST/NRSF alone could cause tumor formation. To determine whether c-Myc and REST/NRSF act together to cause medulloblastomas, we used a …

Translational Regulation Of Nuclear Gene Cox4 Expression By Mitochondrial Content Of Phosphatidylglycerol And Cardiolipin In Saccharomyces Cerevisiae, Xuefeng Su, William Dowhan

Journal Articles

Previous results indicated that translation of four mitochondrion-encoded genes and one nucleus-encoded gene (COX4) is repressed in mutants (pgs1Delta) of Saccharomyces cerevisiae lacking phosphatidylglycerol and cardiolipin. COX4 translation was studied here using a mitochondrially targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5' and 3' untranslated regions (UTRs). Lack of mtGFP expression independent of carbon source and strain background was established to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but was rather caused directly by the lack of phosphatidylglycerol and cardiolipin in mitochondrial membranes. Reintroduction of …

Prime-Boost Vaccination With Plasmid And Adenovirus Gene Vaccines Control Her2/Neu+ Metastatic Breast Cancer In Mice., Xiaoyan Wang, Jian-Ping Wang, Xiao-Mei Rao, Janet E Price, Heshan S Zhou, Lawrence B Lachman

Journal Articles

INTRODUCTION: Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models.

METHODS: The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad …

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

Journal Articles

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 …

Identification And Characterization Of The Herpes Simplex Virus Type 2 Gene Encoding The Essential Capsid Protein Icp32/Vp19c., S P Yei, S I Chowdhury, B M Bhat, A J Conley, W S Wold, W Batterson

Journal Articles

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide …

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

Journal Articles

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

Journal Articles

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 …