DOCSLIB.ORG

Molecular Biology of the Gene Name Chapter 12

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Molecular Biology of the Gene Name Chapter 12 CHAPTER 10: Molecular Biology of the Gene Name ______________ Chapter 12: Biotechnology Introduction A. Viruses infect organisms by 1. binding to receptors on a host’s target cell & injecting viral genetic material into the cell 2. hijacking the host cell’s own molecules and organelles to produce new copies of the virus. B. The host cell is destroyed, and newly replicated viruses are released to continue the infection. C. Viruses are not generally considered alive because they are not cellular and cannot reproduce on their own. D. Because viruses have much less complex structures than cells, they are relatively easy to study at the molecular level. The Structure of the Genetic Material 10.1 SCIENTIFIC DISCOVERY: Experiments showed that DNA is the genetic material 1. Until the 1940s, the case for proteins serving as the genetic material was stronger than the case for DNA. a. Proteins are made from 20 different amino acids b. DNA was known to be made from just four kinds of nucleotides. 2. Studies of bacteria and viruses ushered in the field of molecular biology, the study of heredity at the molecular level, and revealed the role of DNA in heredity. 3. In 1928, Frederick Griffith discovered that a “transforming factor” could be transferred into a bacterial cell. He found that a. when he exposed heat-killed pathogenic bacteria to harmless bacteria, some harmless bacteria were converted to disease-causing bacteria and b. the disease-causing characteristic was inherited by descendants of the transformed cells. 4. In 1952, Alfred Hershey and Martha Chase used bacteriophages to show that DNA is the genetic ma- terial of T2, a virus that infects the bacterium Escherichia coli (E. coli). a. Bacteriophages (or phages for short) are viruses that infect bacterial cells. b. Phages were labeled with radioactive sulfur to detect proteins or radioactive phosphorus to detect DNA. c. Bacteria were infected with either type of labeled phage to determine which substance was injected into cells and which remained outside the infected cell. d. The sulfur-labeled protein stayed with the phages outside the bacterial cell, while the phosphorus- labeled DNA was detected inside cells. e. Cells with phosphorus-labeled DNA produced new bacteriophages with radioactivity in DNA but not in protein. 10.2 DNA and RNA are polymers of nucleotides 1. DNA and RNA are nucleic acids. 2. One of the two strands of DNA is a DNA polynucleotide, a nucleotide polymer (chain). 3. A nucleotide is composed of a nitrogenous base, five-carbon sugar, and phosphate group. a. The nitrogen bases are grouped according to their shape as either pyrimidines or purines. Nucleotides 2 4. The nucleotides are joined to one another by a sugar-phosphate backbone. Deoxyribose (Found in DNA) Ribose (Found in RNA) Phosphate Ester Bond 3 5. Each type of DNA nucleotide has a different nitrogen-containing base: a. adenine (A), cytosine (C), thymine (T), and guanine (G). 6. RNA (ribonucleic acid) is unlike DNA in that it uses the sugar ribose (instead of deoxyribose in DNA) and RNA has the nitrogenous base uracil (U) instead of thymine. 7. Hydrogen Bonds- weak bond between covalently bonded hydrogen and an atom with an unshared pair of electrons a. Hydrogen acts (+) while oxygen or nitrogen acts (-) b. Adenine and Thymine have 2 hydrogen bonds; Cytosine and guanine have 3 hydrogen bonds Label the following: cytosine, guanine, adenine, thymine, phosphate, phosphodiester bond, hy- drogen bond, 3’ end, 5’ end, deoxyribose 10.3 DNA is a double-stranded helix 1. In 1952, after the Hershey-Chase experiment demonstrated that the genetic material was most likely DNA, a race was on to describe the structure of DNA and explain how the structure and properties of DNA can account for its role in heredity. 2. In 1953, James D. Watson and Francis Crick deduced the secondary structure of DNA, using a. X-ray crystallography data of DNA from the work of Rosalind Franklin and Maurice Wilkins b. Erwin Chargaff’s observation that in DNA the amount of adenine was equal to the amount of thy- mine and the amount of guanine was equal to that of cytosine. %A=%T and %G=%C 3. Watson and Crick reported that DNA consisted of two polynucleotide strands wrapped into a double helix. 4 a. The sugar-phosphate backbone is on the outside. b. The nitrogenous bases are perpendicular to the backbone in the interior. c. Specific pairs of bases give the helix a uniform shape. 4. In 1962, the Nobel Prize was awarded to a. James D. Watson, Francis Crick, and Maurice Wilkins. b. Rosalind Franklin probably would have received the prize as well but for her death from cancer in 1958. Nobel Prizes are never awarded posthumously. 5. The Watson-Crick model gave new meaning to the words genes and chromosomes. The genetic in- formation in a chromosome is encoded in the nucleotide sequence of DNA. DNA Replication 10.4 DNA replication depends on specific base pairing 1. In their description of the structure of DNA, Watson and Crick noted that the structure of DNA sug- gests a possible copying mechanism. 2. DNA replication follows a semiconservative model. a. The two DNA strands separate. b. Each strand is used as a pattern to produce a complementary strand, using specific base pairing. c. Each new DNA helix has one old strand with one new strand. 10.5 DNA replication proceeds in two directions at many sites simultaneously 1. DNA replication begins at the origins of replication where a. DNA unwinds at the origin to produce a “bubble,” b. replication proceeds in both directions from the origin, and replication ends when products from the bubbles merge with each other. 2. DNA replication occurs in the 5´ to 3´ direction. a. Replication is continuous on the 3´ to 5´ template. b. Replication is discontinuous on the 5´ to 3´ template, forming short segments (okazaki fragments) 3. Two key proteins are involved in DNA replication. a. DNA ligase joins small fragments into a continuous chain. b. DNA polymerase adds nucleotides to a growing chain and proofreads and corrects improper base pairings. 5 4. DNA polymerases& DNA ligase also repair DNA damaged by harmful radiation & toxic chemicals. 5. DNA replication ensures that all the somatic cells in a multicellular organism carry the same genetic information. The Flow of Genetic Information from DNA to RNA to Protein 10.6 The DNA genotype is expressed as proteins, which provide the molecular basis for phenotypic traits 1. DNA specifies traits by dictating protein synthesis. 2. The molecular chain of command is from DNA in the nucleus to RNA and RNA in the cytoplasm to protein. 3. Transcription is the synthesis of RNA under the direction of DNA. 4. Translation is the synthesis of proteins under the direction of RNA. 5. The connections between genes and proteins a. The initial one gene–one enzyme hypothesis based on studies of inherited metabolic diseases. b. The one gene–one enzyme hypothesis was expanded to include all proteins. c. Most recently, the one gene–one polypeptide hypothesis recognizes that some proteins are com- posed of multiple polypeptides. 10.7 Genetic information written in codons is translated into amino acid sequences - the sequence of nucleotides in DNA provides a code for constructing a protein. 1. Protein construction requires a conversion of a nucleotide sequence to an amino acid sequence. 2. Transcription rewrites the DNA code into RNA, using the same nucleotide “language.” 3. The flow of information from gene to protein is based on a triplet code: The genetic instructions for the amino acid sequence of a polypeptide chain are written in DNA and RNA as a series of nonoverlapping three-base “words” called codons. 4. Translation involves switching from the nucleotide “language” to the amino acid “language.” 5. Each amino acid is specified by one of the 64 codons. 6. Some amino acids have more than one possible codon. 10.8 The genetic code dictates how codons (found on mRNA) are translated into amino acids 1. Characteristics of the genetic code a. Three nucleotides specify one amino acid. b. 61 codons correspond to amino acids. c. AUG codes for methionine and signals the start of transcription. d. 3 “stop” codons signal the end of translation (UAA, UAG, and UGA) 6 2. Table of Codons 10.9 Transcription produces genetic messages in the form of RNA 1. An RNA molecule is transcribed from a DNA template by a process that resembles the partial synthesis of a DNA strand during DNA replication. 2. RNA nucleotides are linked by the transcription enzyme RNA polymerase. 3. Specific sequences of nucleotides along the DNA mark where transcription begins and ends. 4. The “start transcribe” signal is a nucleotide sequence called a promoter. 5. Transcription begins with initiation, as the RNA polymerase attaches to the promoter. 6. During the second phase, elongation, the RNA grows longer. 7. As the RNA peels away, the DNA strands rejoin. 8. Finally, in the third phase, termination, the RNA polymerase reaches a sequence of bases in the DNA template called a terminator, which signals the end of the gene. 9. The polymerase molecule now detaches from the RNA molecule and the gene. 7 10.10 Eukaryotic RNA is processed before leaving the nucleus as mRNA 1. Messenger RNA (mRNA) a. encodes amino acid sequences and conveys genetic messages from DNA to the translation ma- chinery of the cell, which in i. prokaryotes occurs in the same place that mRNA is made ii. eukaryotes mRNA must exit the nucleus via nuclear pores to enter the cytoplasm. c. Eukaryotic mRNA has introns, interrupting sequences that separate while exons, the coding re- gions.
Load more

Recommended publications
  • DNA Recombination Is Sufficient for Retroviral Transduction JODY R
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2460-2464, March 1995 Biochemistry DNA recombination is sufficient for retroviral transduction JODY R. SCHWARTZ, Susi DUESBERG, AND PETER H. DUESBERG Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3206 Contributed by Peter H. Duesberg, November 28, 1994 ABSTRACT Oncogenic retroviruses carry coding se- quences that are transduced from cellular protooncogenes. Nat- env pOl X ural transduction involves two nonhomologous recombinations and is thus extremely rare. Since transduction has never been reproduced experimentall, its mechanism has been studied in terms oftwoehypotheses: (i) the DNAmodel,which postulates two ___ _-I ____ DNA recombinations, and (ii) the RNA model, which postulates a 5' DNA recombination and a 3' RNA recombination occurring during reverse transcription of viral and protooncogene RNA. proto-onc gene Here we use two viral DNA constructs to test the prediction ofthe DNA model that the 3' DNA recombination is achieved by conventional integration of a retroviral DNA 3' of the chromo- somal protooncogene coding region. For the DNA model to be ga-onc - gag viable, such recombinant viruses must be infectious without the w5 essential tract that precedes the 3' purportedly polypurine (ppt) FIG. 1. The DNA model of retroviral transduction. The model long terminal repeat (LTR) of all retroviruses. Our constructs proposes that the 5' retrovirus/protooncogene junction is achieved by consist ofa ras coding region from Harvey sarcoma virus which nonhomologous recombination between a circular provirus with a is naturally linked at the 5' end to a retroviral LTR and single LTR. Such proviruses are common in virus-infected cells (1).
    [Show full text]
  • The LUCA and Its Complex Virome in Another Recent Synthesis, We Examined the Origins of the Replication and Structural Mart Krupovic , Valerian V
    PERSPECTIVES archaea that form several distinct, seemingly unrelated groups16–18. The LUCA and its complex virome In another recent synthesis, we examined the origins of the replication and structural Mart Krupovic , Valerian V. Dolja and Eugene V. Koonin modules of viruses and posited a ‘chimeric’ scenario of virus evolution19. Under this Abstract | The last universal cellular ancestor (LUCA) is the most recent population model, the replication machineries of each of of organisms from which all cellular life on Earth descends. The reconstruction of the four realms derive from the primordial the genome and phenotype of the LUCA is a major challenge in evolutionary pool of genetic elements, whereas the major biology. Given that all life forms are associated with viruses and/or other mobile virion structural proteins were acquired genetic elements, there is no doubt that the LUCA was a host to viruses. Here, by from cellular hosts at different stages of evolution giving rise to bona fide viruses. projecting back in time using the extant distribution of viruses across the two In this Perspective article, we combine primary domains of life, bacteria and archaea, and tracing the evolutionary this recent work with observations on the histories of some key virus genes, we attempt a reconstruction of the LUCA virome. host ranges of viruses in each of the four Even a conservative version of this reconstruction suggests a remarkably complex realms, along with deeper reconstructions virome that already included the main groups of extant viruses of bacteria and of virus evolution, to tentatively infer archaea. We further present evidence of extensive virus evolution antedating the the composition of the virome of the last universal cellular ancestor (LUCA; also LUCA.
    [Show full text]
  • Effects of Retroviruses on Host Genome Function
    ANRV361-GE42-20 ARI 1 August 2008 18:2 V I E E W R S I E N C N A D V A Effects of Retroviruses on Host Genome Function Patric Jern and John M. Coffin Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111; email: [email protected], John.Coffi[email protected] Annu. Rev. Genet. 2008. 42:20.1–20.23 Key Words The Annual Review of Genetics is online at Human Endogenous Retrovirus, LTR, transcription, recombination, genet.annualreviews.org methylation This article’s doi: 10.1146/annurev.genet.42.110807.091501 Abstract Copyright c 2008 by Annual Reviews. For millions of years, retroviral infections have challenged vertebrates, All rights reserved occasionally leading to germline integration and inheritance as ERVs, 0066-4197/08/1201-0001$20.00 genetic parasites whose remnants today constitute some 7% to 8% of the human genome. Although they have had significant evolutionary side effects, it is useful to view ERVs as fossil representatives of retro- viruses extant at the time of their insertion into the germline, not as direct players in the evolutionary process itself. Expression of particu- lar ERVs is associated with several positive physiological functions as well as certain diseases, although their roles in human disease as etio- logical agents, possible contributing factors, or disease markers—well demonstrated in animal models—remain to be established. Here we discuss ERV contributions to host genome structure and function, in- cluding their ability to mediate recombination, and physiological effects on the host transcriptome resulting from their integration, expression, and other events.
    [Show full text]
  • Proviruses with Long-Term Stable Expression Accumulate In
    viruses Article Proviruses with Long-Term Stable Expression Accumulate in Transcriptionally Active Chromatin Close to the Gene Regulatory Elements: Comparison of ASLV-, HIV- and MLV-Derived Vectors Dalibor Miklík 1,2, Filip Šenigl 1 and Jiˇrí Hejnar 1,* 1 Institute of Molecular Genetics, Czech Academy of Sciences, Videnska 1083, CZ-14220 Prague 4, Czech Republic; [email protected] (D.M.); [email protected] (F.S.) 2 Faculty of Science, Charles University, Albertov 6, CZ-12843 Prague 2, Czech Republic * Correspondence: [email protected] Received: 29 January 2018; Accepted: 6 March 2018; Published: 8 March 2018 Abstract: Individual groups of retroviruses and retroviral vectors differ in their integration site preference and interaction with the host genome. Hence, immediately after infection genome-wide distribution of integrated proviruses is non-random. During long-term in vitro or persistent in vivo infection, the genomic position and chromatin environment of the provirus affects its transcriptional activity. Thus, a selection of long-term stably expressed proviruses and elimination of proviruses, which have been gradually silenced by epigenetic mechanisms, helps in the identification of genomic compartments permissive for proviral transcription. We compare here the extent and time course of provirus silencing in single cell clones of the K562 human myeloid lymphoblastoma cell line that have been infected with retroviral reporter vectors derived from avian sarcoma/leukosis virus (ASLV), human immunodeficiency virus type 1 (HIV) and murine leukaemia virus (MLV). While MLV proviruses remain transcriptionally active, ASLV proviruses are prone to rapid silencing. The HIV provirus displays gradual silencing only after an extended time period in culture.
    [Show full text]
  • Frequent Provirus Insertional Mutagenesis of Notchl in Thymomas of MMTVD/Myc Transgenic Mice Suggests a Collaboration of C-Myc and Notctil for Oncogenesis
    Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Frequent provirus insertional mutagenesis of Notchl in thymomas of MMTVD/myc transgenic mice suggests a collaboration of c-myc and Notctil for oncogenesis Luc Girard, l Zaher Hanna, 1,2 Normand Beaulieu, 1 Caroline D. Hoemann, 1 Carole Simard, 1 Christine A. Kozak, 3 and Paul Jolicoeur 1'4'5"6 1Laboratory of Molecular Biology, Clinical Research Institute of Montr4al, Montr4al, Qu~bec, Canada H2W 1R7; 2D4partement de Mddecine et de 4 Microbiologie et d'Immunologie, Universit~ de Montr4al, Montreal, Quebec, Canada H3J 3J7; 3National Institute of Allergy and Infectious Diseases (NIAIDI, National Institutes of Health, Bethesda, Maryland 20892, USA; SDepartment of Experimental Medicine, McGill University, Montr4al, Quebec, Canada, H3G-1A4 The MMTVD/myc transgenic mice spontaneously develop oligoclonal CD4+CD8 + T-ceU tumors. We used provirus insertional mutagenesis in these mice to identify putative collaborators of c-myc. We found that Notchl was mutated in a high proportion (52%) of these tumors. Proviruses were inserted upstream of the exon coding for the transmembrane domain and in both transcriptional orientations. These mutations led to high expression of truncated Notchl RNAs and proteins (86-110 kD). In addition, many Notchl-rearranged tumors showed elevated levels of full-length Notchl transcripts, whereas nearly all showed increased levels of full-length (330-kD) or close to full-length (280-kD) Notchl proteins. The 5' end of the truncated RNAs were determined for some tumors by use of RT-PCR and 5' RACE techniques. Depending on the orientation of the proviruses, viral LTR or cryptic promoters appeared to be utilized, and coding potential began in most cases in the transmembrane domain.
    [Show full text]
  • Lentivirus and Lentiviral Vectors Fact Sheet
    Lentivirus and Lentiviral Vectors Family: Retroviridae Genus: Lentivirus Enveloped Size: ~ 80 - 120 nm in diameter Genome: Two copies of positive-sense ssRNA inside a conical capsid Risk Group: 2 Lentivirus Characteristics Lentivirus (lente-, latin for “slow”) is a group of retroviruses characterized for a long incubation period. They are classified into five serogroups according to the vertebrate hosts they infect: bovine, equine, feline, ovine/caprine and primate. Some examples of lentiviruses are Human (HIV), Simian (SIV) and Feline (FIV) Immunodeficiency Viruses. Lentiviruses can deliver large amounts of genetic information into the DNA of host cells and can integrate in both dividing and non- dividing cells. The viral genome is passed onto daughter cells during division, making it one of the most efficient gene delivery vectors. Most lentiviral vectors are based on the Human Immunodeficiency Virus (HIV), which will be used as a model of lentiviral vector in this fact sheet. Structure of the HIV Virus The structure of HIV is different from that of other retroviruses. HIV is roughly spherical with a diameter of ~120 nm. HIV is composed of two copies of positive ssRNA that code for nine genes enclosed by a conical capsid containing 2,000 copies of the p24 protein. The ssRNA is tightly bound to nucleocapsid proteins, p7, and enzymes needed for the development of the virion: reverse transcriptase (RT), proteases (PR), ribonuclease and integrase (IN). A matrix composed of p17 surrounds the capsid ensuring the integrity of the virion. This, in turn, is surrounded by an envelope composed of two layers of phospholipids taken from the membrane of a human cell when a newly formed virus particle buds from the cell.
    [Show full text]
  • HIV-1 Virus Cycle Replication: a Review of RNA Polymerase II Transcription, Alternative Splicing and Protein Synthesis Corresponding Autor
    HIV-1 virus cycle replication: a review of RNA polymerase II transcription, alternative splicing and protein synthesis Correspondingautor: MiguelRamos-Pascual Abstract HIV virus replication is a time-related process that includes attachment to host cell and fusion, reverse transcription, integration on host cell DNA, transcription and splicing, multiple mRNA transport, protein synthesis, budding and maturation. Focusing on the core steps, RNA polymerase II transcripts in an early stage pre-mRNA containing regulator proteins (i.e nef,tat,rev,vif,vpr,vpu), which are completely spliced by the spliceosome complex (0.9kb and 1.8kb) and exported to the ribosome for protein synthesis. These splicing and export processes are regulated by tat protein, which binds on Trans-activation response (TAR) element, and by rev protein, which binds to the Rev-responsive Element (RRE). As long as these regulators are synthesized, splicing is progressively inhibited (from 4.0kb to 9.0kb) and mRNAs are translated into structural and enzymatic proteins (env, gag-pol). During this RNAPII scanning and splicing, around 40 different multi-cystronic mRNA have been produced. Long-read sequencing has been applied to the HIV-1 virus genome (type HXB2CG) with the HIV.pro software, a fortran 90 code for simulating the virus replication cycle, specially RNAPII transcription, exon/intron splicing and ribosome protein synthesis, including the frameshift at gag/pol gene and the ribosome pause at env gene. All HIV-1 virus proteins have been identified as far as other ORFs. As observed, tat/rev protein regulators have different length depending on the splicing cleavage site: tat protein varies from 224aa to a final state of 72aa, whereas rev protein from 25aa to 27aa, with a maximum of 119aa.
    [Show full text]
  • Identification of Small Endogenous Viral Elements Within Host
    IDENTIFICATION OF SMALL ENDOGENOUS VIRAL ELEMENTS WITHIN HOST GENOMES by Edward C. Davis, Jr. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Computer Science Boise State University May 2016 c 2016 Edward C. Davis, Jr. ALL RIGHTS RESERVED BOISE STATE UNIVERSITY GRADUATE COLLEGE DEFENSE COMMITTEE AND FINAL READING APPROVALS of the thesis submitted by Edward C. Davis, Jr. Thesis Title: Identification of Small Endogenous Viral Elements within Host Genomes Date of Final Oral Examination: 04 March 2016 The following individuals read and discussed the thesis submitted by student Edward C. Davis, Jr., and they evaluated his presentation and response to questions during the final oral examination. They found that the student passed the final oral examination. Timothy Andersen, Ph.D. Chair, Supervisory Committee Amit Jain, Ph.D. Member, Supervisory Committee Gregory Hampikian, Ph.D. Member, Supervisory Committee The final reading approval of the thesis was granted by Timothy Andersen, Ph.D., Chair, Supervisory Committee. The thesis was approved for the Graduate College by John R. Pelton, Ph.D., Dean of the Graduate College. Dedicated to Elaina, Arianna, and Zora. iv ACKNOWLEDGMENTS The author wishes to express gratitude to the members of the supervisory com- mittee for providing guidance and patience. v ABSTRACT A parallel string matching software architecture has been developed (incorpo- rating several algorithms) to identify small genetic sequences in large genomes. En- dogenous viral elements (EVEs) are sequences originating in the genomes of viruses that have become integrated into the chromosomes of sperm or egg cells of infected hosts, and passed to subsequent generations.
    [Show full text]
  • Viral Vectors 101 a Desktop Resource
    Viral Vectors 101 A Desktop Resource Created and Compiled by Addgene www.addgene.org August 2018 (1st Edition) Viral Vectors 101: A Desktop Resource (1st Edition) Viral Vectors 101: A desktop resource This page intentionally left blank. 2 Chapter 1 - What Are Fluorescent Proteins? ViralViral Vectors Vector 101: A Desktop Resource (1st Edition) ViralTHE VectorsHISTORY 101: OFIntroduction FLUORESCENT to this desktop PROTEINS resource (CONT’D)By Tyler J. Ford | July 16, 2018 Dear Reader, If you’ve worked with mammalian cells, it’s likely that you’ve worked with viral vectors. Viral vectors are engineered forms of mammalian viruses that make use of natural viral gene delivery machineries and that are optimized for safety and delivery. These incredibly useful tools enable you to easily deliver genes to mammalian cells and to control gene expression in a variety of ways. Addgene has been distributing viral vectors since nearly its inception in 2004. Since then, our viral Cummulative ready-to-use virus distribution through June 2018. vector collection has grown to include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. To further enable researchers, we started our viral service in 2017. Through this service, we distribute ready-to- use, quality-controlled AAV and lentivirus for direct use in experiments. As you can see in the chart to the left, this service is already very popular and its use has grown exponentially. With this Viral Vectors 101 eBook, we are proud to further expand our viral vector offerings. Within it, you’ll find nearly all of our viral vector educational content in a single downloadable resource.
    [Show full text]
  • Reference Plasmid Phxb2 D Is an HIV-1 Molecular Clone That Exhibits
    bioRxiv preprint doi: https://doi.org/10.1101/611848; this version posted March 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Reference plasmid pHXB2_D is an HIV-1 molecular clone that exhibits identical LTRs and a single integration site indicative of an HIV provirus Alejandro R. Gener1,2,3,4§, Wei Zou5, Brian T. Foley6, Deborah P. Hyink*2, Paul E. Klotman*1,2 1Integrative Molecular and Biomedical Sciences Program, Baylor College of Medicine, Houston, Texas, USA 2Margaret M. and Albert B. Alkek Department of Medicine, Nephrology, Baylor College of Medicine, Houston, Texas, USA 3Department of Genetics, MD Anderson Cancer Center, Houston, Texas, USA 4School of Medicine, Universidad Central del Caribe, Bayamón, Puerto Rico, USA 5Division of Infectious Diseases, the 1st Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China 6Theoretical Biology and Biophysics Group T-6, Los Alamos National Laboratory, Los Alamos, New Mexico, USA *Equal contributions. §Corresponding author: Alejandro R. Gener One Baylor Plaza Mail Stop 710 Houston, Texas, 77030, USA 9045715562 [email protected] ; [email protected] Keywords: HIV-1, reagent verification, nanopore DNA sequencing, provirus, plasmid, sequence variability, resequencing, LTR phasing bioRxiv preprint doi: https://doi.org/10.1101/611848; this version posted March 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 2 1 Abstract 2 Objective: To compare long-read nanopore DNA sequencing (DNA-seq) with short-read 3 sequencing-by-synthesis for sequencing a full-length (e.g., non-deletion, nor reporter) HIV-1 4 model provirus in plasmid pHXB2_D.
    [Show full text]
  • An Introduction to Viral Vectors: Safety Considerations
    An Introduction to Viral Vectors: Safety Considerations Dawn P. Wooley, Ph.D., SM(NRCM), RBP, CBSP Learning Objectives Recognize hazards associated with viral vectors in research and animal testing laboratories. Interpret viral vector modifications pertinent to risk assessment. Understand the difference between gene delivery vectors and viral research vectors. 2 Outline Introduction to Viral Vectors Retroviral & Lentiviral Vectors (+RNA virus) Adeno and Adeno-Assoc. Vectors (DNA virus) Novel (-)RNA virus vectors NIH Guidelines and Other Resources Conclusions 3 Increased Use of Viral Vectors in Research Difficulties in DNA delivery to mammalian cells <50% with traditional transfection methods Up to ~90% with viral vectors Increased knowledge about viral systems Commercialization has made viral vectors more accessible Many new genes identified and cloned (transgenes) Gene therapy 4 5 6 What is a Viral Vector? Viral Vector: A viral genome with deletions in some or all essential genes and possibly insertion of a transgene Plasmid: Small (~2-20 kbp) circular DNA molecules that replicates in bacterial cells independently of the host cell chromosome 7 Molecular Biology Essentials Flow of genetic information Nucleic acid polarity Infectivity of viral genomes Understanding cDNA cis- vs. trans-acting sequences cis (Latin) – on the same side trans (Latin) – across, over, through 8 Genetic flow & nucleic acid polarity Coding DNA Strand (+) 5' 3' 5' 3' 5' 3' 3' 5' Noncoding DNA Strand (-) mRNA (+) RT 3' 5' cDNA(-) Proteins (Copy DNA aka complementary DNA) 3' 5' 3' 5' 5' 3' mRNA (+) ds DNA in plasmid 9 Virology Essentials Replication-defective vs. infectious virus Helper virus vs. helper plasmids Pathogenesis Original disease Disease caused by transgene Mechanisms of cancer Insertional mutagenesis Transduction 10 Viral Vector Design and Production 1 + Vector Helper Cell 2 + Helper Constructs Vector 3 + + Vector Helper Constructs Note: These viruses are replication-defective but still infectious.
    [Show full text]
  • Function of Host Protein Staufen1 in Rabies Virus Replication
    viruses Article Function of Host Protein Staufen1 in Rabies Virus Replication Gaowen Liu †, Congjie Chen †, Ruixian Xu, Ming Yang, Qinqin Han, Binghui Wang, Yuzhu Song *, Xueshan Xia * and Jinyang Zhang * Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China; [email protected] (G.L.); [email protected] (C.C.); [email protected] (R.X.); [email protected] (M.Y.); [email protected] (Q.H.); [email protected] (B.W.) * Correspondence: [email protected] (Y.S.); [email protected] (X.X.); [email protected] (J.Z.); Tel.: +86-871-65939528 (Y.S. & J.Z.) † These authors contributed equally to this work. Abstract: Rabies virus is a highly neurophilic negative-strand RNA virus with high lethality and remains a huge public health problem in developing countries to date. The double-stranded RNA- binding protein Staufen1 (STAU1) has multiple functions in RNA virus replication, transcription, and translation. However, its function in RABV infection and its mechanism of action are not clear. In this study, we investigated the role of host factor STAU1 in RABV infection of SH-SY-5Y cells. Immunofluorescence, TCID50 titers, confocal microscopy, quantitative real-time PCR and Western blotting were carried out to determine the molecular function and subcellular distribution of STAU1 in these cell lines. Expression of STAU1 in SH-SY-5Y cells was down-regulated by RNA interference or up-regulated by transfection of eukaryotic expression vectors. The results showed that N proficiently colocalized with STAU1 in SH-SY-5Y at 36 h post-infection, and the expression level of STAU1 was also proportional to the time of infection.
    [Show full text]