Cosmid Vectors Forrapid Genomic Walking, Restriction Mapping
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Epstein-Barr Virus Recombinants from Overlapping Cosmid Fragments
JOURNAL OF VIROLOGY, Dec. 1993, p. 7298-7306 Vol. 67, No. 12 0022-538X/93/127298-09$02.00/0 Copyright © 1993, American Society for Microbiology Epstein-Barr Virus Recombinants from Overlapping Cosmid Fragments BLAKE TOMKINSON,t ERLE ROBERTSON, RAMANA YALAMANCHILI, RICHARD LONGNECKER,t AND ELLIOT-T KIEFF* Departments of Medicine and Microbiology and Molecular Genetics, Harvard Medical School, 75 Francis Street, Boston, Massachuisetts 02115 Received 7 July 1993/Accepted 18 August 1993 Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. -
New York Science Journal 2016;9(10) 19 Gene Library Ma Hongbao Brookdale University Hospital A
New York Science Journal 2016;9(10) http://www.sciencepub.net/newyork Gene Library Ma Hongbao Brookdale University Hospital and Medical Center, Brooklyn, New York 11212, USA [email protected] Abstract: A gene library is a collection of gene clones that represents the genetic material of an organism. There are different types of gene libraries, including cDNA libraries, genomic libraries and randomized mutant libraries. The applications of these libraries depend on the source of the original DNA fragments. Generally in a gene library each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of bacteria or yeast such that each organism contains on average one construct. As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated. [Ma Hongbao. Gene Library. N Y Sci J 2016;9(10):19-23]. ISSN 1554-0200 (print); ISSN 2375-723X (online). http://www.sciencepub.net/newyork. 4. doi:10.7537/marsnys091016.04. Keywords: gene; library; clone; organism; DNA; molecule; copy A gene library is a collection of gene clones that cDNA libraries are useful in reverse genetics, but they represents the genetic material of an organism. In only represent a very small (less than 1%) portion of molecular biology, a library is a collection of DNA the overall genome in a given organism. The fragments stored and propagated in a population of applications of cDNA libraries include: (1) Discovery micro-organisms through the process of molecular of novel genes; (2) Cloning of full-length cDNA cloning. -
An Essential Gene for Fruiting Body Initiation in the Basidiomycete Coprinopsis Cinerea Is Homologous to Bacterial Cyclopropane Fatty Acid Synthase Genes
Genetics: Published Articles Ahead of Print, published on December 1, 2005 as 10.1534/genetics.105.045542 An essential gene for fruiting body initiation in the basidiomycete Coprinopsis cinerea is homologous to bacterial cyclopropane fatty acid synthase genes Yi Liu*,1, Prayook Srivilai†, Sabine Loos*,2, Markus Aebi*, Ursula Kües† * Institute for Microbiology, ETH Zurich, CH-8093 Zurich, Switzerland † Molecular Wood Biotechnology, Institute for Forest Botany, Georg-August University Göttingen, D-37077 Göttingen, Germany 1 Present address: Laboratorio di Differenziamento Cellulare, Centro di Biotecnologie Avanzate, Largo R. Benzi 10, 16132 Genova, Italy 2 Present address: Hans Knöll Institut Jena e.V., Junior Research Group Bioorganic Synthesis, Beutenbergstr. 11a, D-07745 Jena, Germany Sequence data from this article have been deposited with the EMBL/Genbank Data Libraries under accession nos. AF338438 1 Running title: An essential fruiting initiation gene Key words: hyphal knots, primordia, sexual development, ∆24-sterol-C-methyltransferase, lipid modification Corresponding author: U. Kües, Georg-August University Göttingen, Institute for Forest Botany, Section Molecular Wood Biotechnology, Büsgenweg 2, D-37077 Göttingen, Germany Tel: ++49-551-397024 Fax: ++49-551-392705 e-mail: [email protected] 2 ABSTRACT The self-compatible Coprinopsis cinerea homokaryon AmutBmut produces fruiting bodies without prior mating to another strain. Early stages of fruiting body development include the dark- dependent formation of primary hyphal knots and their light-induced transition to the more compact secondary hyphal knots. The AmutBmut UV mutant 6-031 forms primary hyphal knots, but development arrests at the transition state by a recessive defect in the cfs1 gene, isolated from a cosmid library by mutant complementation. -
Library Construction and Screening
Library construction and screening • A gene library is a collection of different DNA sequences from an organism, • which has beenAlso called genomic libraries or gene banks. • cloned into a vector for ease of purification, storage and analysis. Uses of gene libraries • To obtain the sequences of genes for analysis, amplification, cloning, and expression. • Once the sequence is known probes, primers, etc. can be synthesized for further diagnostic work using, for example, hybridization reactions, blots and PCR. • Knowledge of a gene sequence also offers the possibility of gene therapy. • Also, gene expression can be used to synthesize a product in particular host cells, e.g. synthesis of human gene products in prokaryotic cells. two types of gene library depending upon the source of the DNA used. 1.genomic library. 2.cDNA library Types of GENE library: • genomic library contains DNA fragments representing the entire genome of an organism. • cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell. Genomic Library : • Made from nuclear DNA of an organism or species. • DNA is cut into clonable size pieces as randomly possible using restriction endonuclease • Genomic libraries contain whole genomic fragments including gene exons and introns, gene promoters, intragenic DNA,origins of replication, etc Construction of Genomic Libraries 1. Isolation of genomic DNA and vector. 2.Cleavage of Genomic DNA and vector by Restriction Endonucleases. 3.Ligation of fragmented DNA with the vector. 4.Transformation of -
Construction of Small-Insert Genomic DNA Libraries Highly Enriched
Proc. Natl. Acad. Sci. USA Vol. 89, pp. 3419-3423, April 1992 Genetics Construction of small-insert genomic DNA libraries highly enriched for microsatellite repeat sequences (marker-selected libraries/CA repeats/sequence-tagged sites/genetic mapping/dog genome) ELAINE A. OSTRANDER*tt, PAM M. JONG*t, JASPER RINE*t, AND GEOFFREY DUYKt§ *Department of Molecular and Cellular Biology, 401 Barker Hall, University of California, Berkeley, CA 94720; tHuman Genome Center, Lawrence Berkeley Laboratory, 1 Cyclotron Road, 74-157, Berkeley, CA 94720; and §Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115 Communicated by Philip Leder, January 8, 1992 ABSTRACT We describe an efficient method for the con- Generation of a high-density map of markers for an entire struction of small-insert genomic libraries enriched for highly genome or a single chromosome requires the isolation and polymorphic, simple sequence repeats. With this approach, characterization of hundreds of markers such as microsatel- libraries in which 40-50% of the members contain (CA). lite repeats (10, 11). Two simple yet tedious approaches have repeats are produced, representing an =50-fold enrichment generally been used for this task. One approach is to screen over conventional small-insert genomic DNA libraries. Briefly, a large-insert genomic library with an end-labeled (CA),, or a genomic library with an average insert size of less than 500 (TG),, oligonucleotide (n > 15). Clones that hybridize to the base pairs was constructed in a phagemid vector. Ampliflcation probe are purified and divided into subclones, which are of this library in a dut ung strain ofEscherchia coli allowed the screened by hybridization for a fragment containing the recovery of the library as closed circular single-stranded DNA repeat. -
Multiple Trans-Splicing Events Are Required to Produce a Mature Nadl Transcript in a Plant Mitochondrion
Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Multiple trans-splicing events are required to produce a mature nadl transcript in a plant mitochondrion Patricia L. Conklin, Robin K. Wilson, and Maureen R. Hanson Section of Genetics and Development, Cornell University, Ithaca, New York 14853 USA The mitochondrial gene encoding NADH dehydrogenase subunit 1 (had1) in Petunia hybrida is split into five exons, a, b, c, d, and e. With the use of a complete restriction map of the 443-kb Petunia mitochondrial genome, we have cloned these exons and mapped their location. Exon a is located 130 kb away from and in the opposite orientation from exons b and c. Exon d maps 95 kb away and in the opposite orientation from exons b and c. Exons d and e are separated by 190 kb. By performing the polymerase chain reaction on Petunia cDNAs, we have shown that transcripts from these five exons are joined via a series of cis- and trans-splicing events to create a mature had1 transcript. In addition, we have found 23 C -* U RNA edit sites in Petunia nadl. RNA editing changes 19 of the amino acids predicted by the genomic sequence. [Key Words: trans-splicing; nadl ; RNA editing; mitochondria; Petunia hybrida; introns] Received April 29, 1991; revised version accepted May 31, 1991. Several different RNA processing events can occur dur- NADH-dehydrogenase subunit I (had1) also contains in- ing the maturation of a protein-coding plant mitochon- trons. In contrast, nadl is encoded by a single open read- drial transcript. -
TI-Pos321 T'u-Pos323 and CHOLESTEROL. ((A.C
iismalanlr AvWDMR rxunnbP12^R1;| qjrAI bCrDTBTVWTTTI-MuIL-1 UKM AA"ANVn nTVAMT.4v I NAMAlb AAUIAini TI-Pos321 Ib-Pos322 Dithionite Quenching Rate Measurement of the Inside-Outside MEASUREMENT OF FLUORESCENCE SIGNAL OF A Membrane Bilayer Distribution of NBD-Labeled Phospholipids ((Cesar VOLTAGE-SENSITIVE DYE USING TWO-PHOTON EXCITATION Angeletti and J. Wylie Nichols.)) Department of Physiology. Emory University ((S.T. Hess, W.W. Webb.)) NIH Developmental Resource for Biophysical Imaging School of MIedicine. Atlanta GA 30322 and Opto-electronics, Cornell University, Ithaca, NY 14853 Dithionite quenching of the fluorescence of 'NBD-labeled lipids has been used to Fluorescent voltage-sensitive dyes have the potential for great utility in neuro- quantify their distribution and translocation across cellular and organellar mem- science if the sensitivity, i.e. the fractional change in fluorescence intensity (AF/F) branes. Assaying the extent of dithionite quenching provides a straightforward for a 100 mV depolarization, can be improved. We have measured the two-photon method for determining the fraction of NBD-lipid exposed to the outer leaflet of excitation cross-section for di-8-ANEPPS, a particularly bright, photostable styryl- membranes that are impermeant to dithionite. However. it appears that many. if class dye, using a pulsed Ti:Sapphire infared laser, and have chosen an appropriate not all, cellular membranes are relatively permeable to dithionite. The present work wavelength for excitation in cells. Using two-photon excitation, bright fluorescence describes a method in which the initial rate of dithionite quenching. rather than the was observed in HeLa cells. Preliminary results show a large AF/F (30-50%) upon extent of quenching, was used to determine the fraction of different NBD-labeled depolarization of the cells using 200 mM KCI. -
Molecular Biology and Applied Genetics
MOLECULAR BIOLOGY AND APPLIED GENETICS FOR Medical Laboratory Technology Students Upgraded Lecture Note Series Mohammed Awole Adem Jimma University MOLECULAR BIOLOGY AND APPLIED GENETICS For Medical Laboratory Technician Students Lecture Note Series Mohammed Awole Adem Upgraded - 2006 In collaboration with The Carter Center (EPHTI) and The Federal Democratic Republic of Ethiopia Ministry of Education and Ministry of Health Jimma University PREFACE The problem faced today in the learning and teaching of Applied Genetics and Molecular Biology for laboratory technologists in universities, colleges andhealth institutions primarily from the unavailability of textbooks that focus on the needs of Ethiopian students. This lecture note has been prepared with the primary aim of alleviating the problems encountered in the teaching of Medical Applied Genetics and Molecular Biology course and in minimizing discrepancies prevailing among the different teaching and training health institutions. It can also be used in teaching any introductory course on medical Applied Genetics and Molecular Biology and as a reference material. This lecture note is specifically designed for medical laboratory technologists, and includes only those areas of molecular cell biology and Applied Genetics relevant to degree-level understanding of modern laboratory technology. Since genetics is prerequisite course to molecular biology, the lecture note starts with Genetics i followed by Molecular Biology. It provides students with molecular background to enable them to understand and critically analyze recent advances in laboratory sciences. Finally, it contains a glossary, which summarizes important terminologies used in the text. Each chapter begins by specific learning objectives and at the end of each chapter review questions are also included. -
Adenovirus Expression Vector Kit (Dual Version)
Ver.0.90 Code No. 6170 Adenovirus Expression Vector Kit (Dual Version) - 1 - Ver.0.90 Precautions for the use of this product ・ Please follow the guideline for experiments using recombinant DNA issued by the relevant authorities and the safety committee of your organization or your country in using this product. ・ The use of this product is limited for research purposes. It must not be used for clinical purposes or for in vitro diagnosis. ・ Individual license agreement must be concluded when this product is used for industrial purposes. ・ In this kit, recombinant viral particles infectious to mammalian cells are produced in 293 cells. Although this recombinant virus cannot proliferate in cells other than 293 cells, in case that it attaches to the skin or the airway, it efficiently enters cells and expresses the target gene. Please use a safety cabinet to prevent inhalation or adhesion of the virus. ・ Basic techniques of genetic engineering and cell cultivation are needed for the use of this product. ・ The user is strongly advised not to generate recombinant adenovirus capable of expressing known oncogenes and any genes known to be hazardous to the mammals. ・ Takara is not liable for any accident or damage caused by the use of this product. - 2 - Ver.0.90 Table of ContentsContents ⅠⅠ.. Introduction Ⅰ-1. Adenovirus vectors ……………………………………….…………………... 5 Ⅰ-2. Product overview ……………………………………………………………... 5 ⅡⅡ.. General Information …………………………………………………………….. 7 ■ For Adenovirus Expression Vector Kit (previous version) users …..……………… 8 ⅢⅢ.. Principles of recombinant adenovirus preparation Ⅲ-1. Full-length DNA transfer method ………………………………......... 9 Ⅲ-2. COS-TPC method ……………………………................................…...... 9 ⅣⅣ.. Content of the kit Ⅳ-1. Kit Component ……………………………………………… 12 Ⅳ-2. Cosmid vector structures ……………………………………………………… 13 ⅤⅤ.. Instruments and reagents required other than the kit Ⅴ-1. -
Cdna Libraries and Expression Libraries
Solutions for Practice Problems for Recombinant DNA, Session 4: cDNA Libraries and Expression Libraries Question 1 In a hypothetical scenario you wake up one morning to your roommate exclaiming about her sudden hair growth. She has been supplementing her diet with a strange new fungus purchased at the local farmer’s market. You take samples of the fungus to your lab and you find that this fungus does indeed make a protein (the harE protein) that stimulates hair growth. You construct a fungal genomic DNA library in E. Coli with the hope of cloning the harE gene. If you succeed you will be a billionaire! You obtain DNA from the fungus, digest it with a restriction enzyme, and clone it into a vector. a) What features must be present on your plasmid that will allow you to use this as a cloning vector to make fungal genomic DNA library. Your vector would certainly need to have a unique restriction enzyme site, a selectable marker such as the ampicillin resistance gene, and a bacterial origin of replication. Other features may be required depending upon how you plan to use this library. b) You clone your digested genomic DNA into this vector. The E. coli (bacteria) cells that you will transform to create your library will have what phenotype prior to transformation? Prior to transformation, the E. coli cells that you will transform will be sensitive to antibiotic. This allows you to select for cells that obtained a plasmid. c) How do you distinguish bacterial cells that carry a vector from those that do not? Cells that obtained a vector will have obtained the selectable marker (one example is the ampicillin resistance gene). -
Introduction to Viroids and Prions
Harriet Wilson, Lecture Notes Bio. Sci. 4 - Microbiology Sierra College Introduction to Viroids and Prions Viroids – Viroids are plant pathogens made up of short, circular, single-stranded RNA molecules (usually around 246-375 bases in length) that are not surrounded by a protein coat. They have internal base-pairs that cause the formation of folded, three-dimensional, rod-like shapes. Viroids apparently do not code for any polypeptides (proteins), but do cause a variety of disease symptoms in plants. The mechanism for viroid replication is not thoroughly understood, but is apparently dependent on plant enzymes. Some evidence suggests they are related to introns, and that they may also infect animals. Disease processes may involve RNA-interference or activities similar to those involving mi-RNA. Prions – Prions are proteinaceous infectious particles, associated with a number of disease conditions such as Scrapie in sheep, Bovine Spongiform Encephalopathy (BSE) or Mad Cow Disease in cattle, Chronic Wasting Disease (CWD) in wild ungulates such as muledeer and elk, and diseases in humans including Creutzfeld-Jacob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Alpers syndrome (in infants), Fatal Familial Insomnia (FFI) and Kuru. These diseases are characterized by loss of motor control, dementia, paralysis, wasting and eventually death. Prions can be transmitted through ingestion, tissue transplantation, and through the use of comtaminated surgical instruments, but can also be transmitted from one generation to the next genetically. This is because prion proteins are encoded by genes normally existing within the brain cells of various animals. Disease is caused by the conversion of normal cell proteins (glycoproteins) into prion proteins. -
Clusters of Alpha Satellite on Human Chromosome 21 Are Dispersed Far Onto the Short Arm and Lack Ancient Layers
Loyola University Chicago Loyola eCommons Bioinformatics Faculty Publications Faculty Publications 2016 Clusters of Alpha Satellite on Human Chromosome 21 Are Dispersed Far onto the Short Arm and Lack Ancient Layers William Ziccardi Loyola University Chicago Chongjian Zhao Loyola University Chicago Valery Shepelev Russian Academy of Sciences Lev Uralsky Russian Academy of Sciences Ivan Alexandrov Russian Academy of Medical Sciences Follow this and additional works at: https://ecommons.luc.edu/bioinformatics_facpub See next page for additional authors Part of the Bioinformatics Commons, Evolution Commons, and the Genetics Commons Recommended Citation Ziccardi, William; Zhao, Chongjian; Shepelev, Valery; Uralsky, Lev; Alexandrov, Ivan; Andreeva, Tatyana; Rogaev, Evgeny; Bun, Christopher; Miller, Emily; Putonti, Catherine; and Doering, Jeffrey. Clusters of Alpha Satellite on Human Chromosome 21 Are Dispersed Far onto the Short Arm and Lack Ancient Layers. Chromosome Research, 24, : 421-436, 2016. Retrieved from Loyola eCommons, Bioinformatics Faculty Publications, http://dx.doi.org/10.1007/s10577-016-9530-z This Article is brought to you for free and open access by the Faculty Publications at Loyola eCommons. It has been accepted for inclusion in Bioinformatics Faculty Publications by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. © Springer, 2016. Authors William Ziccardi, Chongjian Zhao, Valery Shepelev, Lev Uralsky, Ivan Alexandrov, Tatyana Andreeva, Evgeny Rogaev, Christopher Bun, Emily Miller, Catherine Putonti, and Jeffrey Doering This article is available at Loyola eCommons: https://ecommons.luc.edu/bioinformatics_facpub/29 Abstract Click here to download Abstract ABSTRACT.docx ABSTRACT Human alpha satellite (AS) sequence domains that currently function as centromeres are typically flanked by layers of evolutionarily older AS that presumably represent the remnants of earlier primate centromeres.