The University of the West Indies,
at Mona

Development and Differentiation

Introduction to Molecular Medicine

Table of Contents

• Introduction
• How to use this guide
• Assessment
• Evaluations
• Aims
• Content
• Learning Objectives
• Teaching Method
• Resources
• Lecture Titles
• Glossary
• Self Assessment Questions
• Answers and Feedback

Introduction

This module is the second of three which comprises the Development and Differentiation Course and is taught in Semester 1 of the first year. It covers medical aspects of genetics including population genetics. Molecular tech­niques used in diagnosis and treatment are presented and ethical implica­tions surrounding the application of molecular biology to medicine are also introduced.

The teaching staff is drawn primarily from the Section of Biochemistry and the Department of Microbiology.

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How to use this guide

Read through the guide at least once to give you an idea of the format of the module. The ‘Aims ’ should be useful to assist you in defining the areas that need to be covered. Look at the lecture titles prior to attending lectures. They will give you a better idea of the upcoming topics and allow you the opportunity to do some preparatory reading.

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Assessment

In this module, students will be assessed by a combination of in­course work and by inclusion of questions on the materials in the integrated end­of­semester examination. In­course assessment will include at least one course test and grading of a literature review assignment.

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Evaluation

The Curriculum Committee is particularly interested in finding out the views of students about their experience in the MBBS curriculum. A Semester 1 course evaluation form will be given to you near the end of the semester. The committee would appreciate a few minutes of your time to fill in this form, as the feedback will be used to assist in maintaining and improving the MBBS programme.

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Aims

The aim of the module is to introduce students to the principles of Molecular Biology, and how it is used to understand and treat human disease. It builds on the fundamentals of the structure and basic functions of nucleic acids and proteins, covered in the Cell Biology module and will serve as a foundation for other topics in the Development and Differentiation Course.

The areas that will be covered include:

1. DNA, RNA structure, chromosomes, genes, genetic code, gene expression, mutations, genome diversity, laboratory manipulation of nucleic acids
2. How genes are inherited, population genetics
3. Molecular techniques, emphasizing those used in diagnosis and treatment
4. Molecular medicine in practice – recombinant proteins, gene therapy, pharmacogenomics, the Human Genome Project (HGP).
5. Ethical considerations and implications in the application of molecular biology to medicine.

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Content

More specifically, this module will focus on the basic principles of:

• DNA as genetic material, double helix, genetic code, ‘central dogma’
• Genome diversity – bacterial, viral and retroviral genomes, plasmids, extra­chromosomal DNA
• DNA replication , transcription
• Translation/protein synthesis ­splicing in haemoglobin and immunoglobulin synthesis, post translational modifications
• Antibiotic action on bacterial replication and protein synthesis
• Population genetics: inheritance, linkage
• Mutations: focus on haemoglobinopathies
• In­born errors of metabolism
• DNA and RNA isolation
• Chromosome karyotyping, in situ hybridization
• Recombinant DNA technology – hybridization, Polymerase Chain Reaction (PCR), trangenesis
• Production of recombinant proteins, monoclonal antibodies
• The Human Genome Project (HGP) – applications, ethical issues
• Gene therapy, pharmacogenomics

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Learning Objectives

At the end of this module, the student should be able to:

• describe how DNA was shown to be the genetic material
• describe how DNA replicates itself and how mutations may arise
• explain how the information carried in DNA is translated into synthesis of proteins
• explain antibiotic action at the level of replication and protein synthesis
• state the ‘central’ dogma of molecular biology
• explain how some viruses do not conform to this dogma
• explain how some retroviruses may cause disease
• describe how mutations may lead to disease e.g. sickle cell, cystic fibro­sis, thalassemias
• compare and contrast the genomes of prokaryotes and humans and dis­cuss the diversity in relation to disease and treatment
• describe methods for isolation and purification of DNA, hybridization
• describe the techniques of PCR, Restriction Fragment Length Polymorphism (RFLP), sequencing, DNA fingerprinting, transgenesis and in situ hybridization
• relate these techniques to their uses in the diagnosis of genetic and infec­tious diseases and in Forensic Medicine
• describe the use of molecular biology techniques in the production of recombinant proteins and antibodies
• provide an overview of the Human Genome Project (HGP)
• describe the fundamentals of somatic gene therapy
• discuss the ethical issues associated with these techniques, the HGP, gene therapy and pharmacogenomics

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Teaching Methods

These will include lectures, tutorials, practical laboratory sessions, visits to laboratories and computer based sessions.

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Resources

Textbook of Biochemistry with Clinical Correlations. Fourth edition. Editor T. M. Devlin

Useful Websites

1. www.ncbi.nlm.nih.gov/genome/central
2. http://www. ebi.ac.uk
3. http://www.ddbj.nig.ac.jp

Prepared handouts

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Lecture Titles

Session	Subject Matter	Resource
1	Nucleic Acid & DNA structure	Dr. Ashby
2	Genome Diversity	Dr. Ashby
3	DNA Replication	Dr.Ashby
4	Transcription	Dr.Anderson­McFarlane
5	Transcription & Protein Synthesis	Dr.Anderson­McFarlane
6	Regulation of Gene Expression	Dr.Anderson­McFarlane
7	Molecular Techniques: Karyotyping	Dr. Anderson­McFarlane
8	Molecular Techniques: Blotting etc.	Dr.Anderson­McFarlane
9	Molecular Techniques: PCR	Dr.Anderson­McFarlane
10	Molecular Techniques: Transgenesis	Dr.Anderson­McFarlane
11	Mutations	Dr. McKenzie
12	Haemoglobinopathies & Cystic Fibrosis	Dr. McKenzie
13	Multifactional diseases	Dr. McKenzie
14	Genetic Inheritance	Dr. McKenzie
15	Population Genetics	Dr. McKenzie
16	Biotechnology: Recombinant proteins	Dr.Anderson­McFarlane
17	Human Genome Project	Dr.Anderson­McFarlane
18	Gene Therapy	Dr.Anderson­McFarlane

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Glossary 9

Terms frequently encountered:

• Allele ­ One of the different forms of a gene that can exist at a single locus on a chromosome.
• Autosomes ­ All the chromosomes except the sex chromosomes; found in somatic cells.
• cDNA ­ A single–stranded DNA complementary to an RNA from which it was synthesized using reverse transcriptase.
• Clone ­ A large number of cells or molecules derived from a single ancestral cell or molecule.
• Denaturation ­ Conversion from the double­stranded to the single­strand­ed state, most often accomplished by heat
• Diploid set of chromosomes ­Contains two copies of each autosome and two sex chromosomes.
• DNA (deoxyribonucleic acid) ­ A double chain of linked nucleotides (having deoxyribose as their sugars); the fundamental unit of which genes are composed.
• Eukaryotic cell ­ A cell containing a nucleus.
• Exons ­ DNA sequences that are transcribed into protein structures.
• Gene therapy ­ Introduction of a fully functional and expressible gene into a target cell, aimed at correcting a specific disease permanently.
• Gene ­ Fundamental physical and functional unit of heredity; a segment of DNA that makes transcription possible.
• Genetic code ­ The correspondence between triplets in DNA (or RNA) and amino acids in protein.
• Genome ­ The complement of genetic information unique to each species of organism.
• Haploid set of chromosomes ­ One copy of each autosome and one sex chromosome; characteristic of germ cells.
• Heterozygote ­ An individual with different alleles of a particular gene, e.g. HbAS (sickle cell trait).
• Histone ­ A type of basic protein that forms a unit around which DNA is tightly coiled in the nucleosomes of eukaryotic chromosomes.
• HomozygoteAn individual with identical alleles for a particular gene, e.g. HbAA (normal), HbSS (sickle cell disease).
• Hybridization Variety of related techniques based on observation that two single­stranded nucleic acids of complementary base sequences will form a double­stranded hybrid.
• Hyperchromicity ­ The increase in optical density that occurs when DNA is denatured.
• Introns Region of a genes ;DNA that is not translated into aprotein.
• Karyotyp The entire chromosomal complement of cell or species (as visualized ;during mitosis).
• Linkage ­ The association of genes on the same chromosome.
• Linkage disequilibrium A situation in which some combinations of genetic markers occur more or less frequently in the population than would be expected from their distance apart.
• Locus ­ Chromosomal ;location of a gene or other piece of DNA.
• Marker allele;Any allele of interest in an experiment.
• Marker DNA ;Fragment of known size used to calibrate an electrophoretic
• mRNA (messenger RNA) ­ An RNA molecule transcribed from the DNA of a gene, and from which a protein is translated by the action of ribo­somes.
• Mutation ­ A process that produces a gene or chromosome set different from that of the wild type. The result of such a process.
• Nucleases Enzymes ;that can degrade ;nucleic acids by breaking the phos­phodiester bonds.
• Nucleotide A molecule composed of a base, a sugar and a phosphate group; the basic building block of nucleic acids.
• Polymorphism A variation in DNA sequence within a population.
• Prokaryotic ;cell ­ A cell with no nuclear membrane, hence, no separate nucleus.
• Restriction enzyme A bacterial endonuclease that recognizes specific base sequences in DNA and breaks the DNA chain at those points.
• Ribosomes Complex of protein and ribosomal RNA (rRNA) that catalyzes the translation of mRNA into an amino acid sequence
• RNA (ribonucleic acid) A single stranded nucleic acid (may fold back on itself to form double­stranded regions), having ribose as sugar and uracil rather than thymine as one of its bases.
• Splicing The removal of introns and joining of exons in RNA; introns are spliced out and exons are spliced together.
• tRNA A class of small RNA; molecules that take specific amino acids to the ribosome during translation such that amino acids are inserted to form a polypeptide chain
• Wild type The genotype or phenotype found in nature or in a standard laboratory stock

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Self­Assessment Questions

Indicate which one of the following is true:

1. The 4 nucleosides found in DNA are:
   1. Adenine, thymine, cytosine, uracil
   2. Adenosine, guanine, cytosine, taurine
   3. denosine, guanosine, thymidine, cytidine
   4. Adenine, thymidine, deoxythymidine, doexyguanosine
2. The amino acid glutamic acid is replaced by valine in:
   1. ß­thalassemia
   2. pernicious anaemia
   3. sickle cell disease
   4. polycythaemia vera
3. The classic PCR technique (without allele specific primers and probes) can be used to:
   1. amplify the intact dystrophin gene (79 exons + 79 introns) in one piece
   2. diagnose the sickle cell mutation after amniocentesis
   3. diagnose HIV infection in people with ‘at risk’ lifestyles
   4. detect point mutations in a gene of unknown sequence
4. DNA restriction fragments are usually separated one from the other by:
   1. Paper chromatography
   2. Electrophoresis in agarose gels
   3. High­speed centrifugation
   4. High­performance liquid chromatography
5. An inhibitor of reverse transcriptase would be useful in:
   1. preventing lytic infection by T4 bacteriophage
   2. curing rabies, a disease caused by an RNA virus
   3. curing AIDS, a disease caused by a retrovirus
   4. inhibiting homologous recombination between two DNAs
6. Some patients with sickle cell disease have relatively mild symptoms because they also have
   1. bone marrow depression
   2. haemoglobin H
   3. increased a­chain synthesis
   4. increased g­chain synthesis
7. All of the following are true except:
   1. Retroviral vectors are more popular than other viruses for somatic gene therapy because they can integrate themselves into host­cell DNA.
   2. A constriction appears in the X chromosome of individuals who have symptoms of Down syndrome.
   3. Less than 1% of our genomes contribute to our individuality.
   4. Virtually all ailments (except trauma) have some genetic basis.

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Answers and Feedback

1. (c) Nucleosides, i.e the nitrogenous bases adenine, cytosine, guanine, thymine bound to deoxyribose are found in DNA
2. (c) Sickle cell disease is the result of an A Æ T transversion in the b­glo­bin chain, GAG Æ GTG (glu Æ val)
3. (c) PCR, without the use of allele specific primers and probes, is used to amplify short segments of DNA. Primers are designed based on knowledge of the DNA sequence
4. (b) DNA fragments travel towards the anode in gel electrophoresis and their positions on the gel are based on size, the smaller fragments move faster
5. (c) A retrovirus needs a reverse transcriptase to make DNA copies of its RNA genome for insertion into the host genome.
6. (d) A high level of HbF ( gchains) is protective in all b–chain abnormal­ities
7. (b) This constriction is seen in cells from individuals with symptoms of fragile X syndrome. Trisomy 21 is found in Down syndrome.

Introduction to Molecular Medicine 15 16 Introduction to Molecular Medicine

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