Molecular Genetics

Code:

MI072111

Acronym:

GENMOL

Keywords
Classification	Keyword
OFICIAL	Natural Sciences

Instance: 2019/2020 - 1S

Active?	Yes
Responsible unit:	Biochemistry Laboratory
Course/CS Responsible:	MSc in Pharmaceutical Sciences

Cycles of Study/Courses

Acronym	No. of Students	Study Plan	Curricular Years	Credits UCN	Credits ECTS	Contact hours	Total Time
MICF	253	Official Curriculum	2	-	6	65	162

Teaching language

Suitable for English-speaking students

Objectives

Study of the basic mechanisms that allow the genetic DNA transmission based in replication, transcription, processing and translation processes. Modifications at DNA level analysis after recombination, mutation, transposition, transduction and viruses. Study of DNA repair mechanisms. Prokariotic and eukariotic gene expression regulation analysis to understand how gene expression is controlled and cells synthesize the right proteins at the right time in the right amounts. Study of the molecular mechanisms associated with cancer and related processes that involve the cell cycle regulation, mutations in proto-oncogenes and anti-oncogenes. The use of DNA replication technology as a dominant approach for studying the basic biological processes that allow cloning and identification of genes, analysis of genetic expression and regulation, diseases diagnosis, production of drugs and vaccines, and gene therapy.

Learning outcomes and competences

The advances that occur in Genetics, including the progress of scientific research, international engagement in mapping the human genome project (HGP) and development of specific techniques are crucial for the assessment of physiological and pathological systems in various areas of health but equally relevant for agriculture, chemical, electronics, energy, toxicology, pharmaceutical and forensic sciences. The knowledge acquired in this UC could be applied to several UCs which are then taught in the curriculum of the MICF, giving powers to the pharmacist for developing and implementing in various areas of work. The execution of laboratory work is done with the aim of carrying out a specific methodology, to contact directly with organic products, materials, reagents and instruments, as well as consolidate the improvement of scientific concepts.

 

Working method

Presencial

Pre-requirements (prior knowledge) and co-requirements (common knowledge)

Are required prior knowledge of Cell Biology, General Microbiology and Biochemistry and simultaneous knowledge of Organic Chemistry and Immunology.

Program

PROGRAM OF THEORETICAL CLASSES:

DNA structure: DNA Discovery; Composition and structure of DNA; Gene, exons, introns, regulatory sequences upstream and downstream; palindromes; Denaturation and renaturation of DNA; DNA compaction; nucleosome; Chromatin and chromosomes; karyotype; Types of DNA: Simple transcription units and complex; unique sequences; moderately repetitive sequences: mobile DNA elements (insertion sequences, transposons compounds and not compounds, LTR retrotransposons and non-LTR; simple and replicative transposition, very repetitive sequences: satellite DNA, microsatellite and minisatellite; pseudogenes processed and unprocessed.

Genetic code: Features; wobble concept. the genetic code in mitochondria.

DNA Replication: The central dogma of molecular biology; Replication in prokaryotes and eukaryotes: origin of replication, required enzymes and their functions DNA polymerases. Primases, ribonucleases; topoisomerases, ligases, helicases, Klenow. Telomeres and Telomerase, shelterina complex; diseases associated with defects in the replication replication types: Type eye; rolling circle type ( "rolling circles"); "D-loop"; Replication of the single-stranded M13 phage.

Transcription: Transcription Regulation; RNA synthesis in prokaryotes; RNA polymerases of eukaryotes and their functions; characteristic of the promoters in eukaryotes and prokaryotes; Sense RNA and antisense RNA; initiation complex in prokaryotes and eukaryotes

Types of RNA: mRNA, tRNA, rRNA, snRNA, snoRNA, scRNA, miRNA and siRNA. MRNA: mRNA in prokaryotes and in eukaryotic mRNA. Shine-Dalgarno sequence and CAP structure; Transfer RNA. Ribosomal RNA: rRNA prokaryote and eukaryote rRNA.

Processing or tRNA splicing; of rRNAs; miRNAs and mRNAs. Splicing mitochondria. RNA "editing". Polyadenylation. Splicing and alternative polyadenylation. Mirtrões and RNA interference mechanisms (RNAi). Pseudogenes.

Translation and protein synthesis: Different stages of translation: Initiation; Elongation and termination. Protein location in prokaryotes and eukaryotes.

Genetic Recombination: Homologous recombination in E. coli: recA, RecBCD; Homologous recombination in eukaryotes: complex Rad, BRCA1 and BRCA2; Holliday structures, heteroduplexes and migration arms; gene conversion; Recombination specific location; Recombination by transposition, Complementation.

Mapping: Maps: genetic, physical, chromosomal or cytogenetic, connection. map unit. Loci. Determination of the distance between genes.

Genetics of bacteria: bacterial chromosome; auxotrophic and prototrophic bacteria. Mutations in E. coli; Enrichment mutants: direct selection, counter-selection; Physical selection; Genetic recombination in E. coli combination; transduction; transformation; transposition.

Phage Genetics: Structure; Infection: lytic and lysogenic cycle; Plaques; concatamers; Replication and Mapping.

Virus: Structure; virus classification; Entry and exit of the virus in the cell; Replication strategies in classes I, II, III, IV, V, VI and VII: main features. life cycle of HIV-1 virus. Viroids. Prions in humans and animals.

DNA mutation: mutagenic agents. mutagenesis mechanisms: specific mismatch, incorporating similar bases, loss of annealing specificity. spontaneous and induced mutations; "Frameshift" mutations; Point mutations: Transition and transversion; nonsense mutations ( "nonsense"); nonsense mutations ( "missense"); silent mutations; neutral mutations; Mutations by transposition; reversible changes; suppressor mutations and ribosomal mutations. Errors caused by streptomycin. Identification mutagens - Ames test: mice with bacteria and enzymes.

DNA repair in prokaryotes and mammals: photoreactivation; Removal direct alkylation damage; Error prevention; Excision repair: general and specific system in prokaryotes; BER system in mammals; translesion mechanism in prokaryotes and eukaryotes; post-replication repair: mismatch repair, by recombination by induction of the SOS genes; Repair prone to errors (bypass synthesis); Repair the GO system; associated transcription repair (TCR). BER and NER. Repair by joining the non-homologous ends. Repair by intragenic and extragenic. Repair by mutant t-RNAs. Diseases associated with defects in the repair mechanisms.

Recombinant DNA techniques: Molecular genetics and reverse genetics. cloning concept; Restriction enzymes; Modifications enzymes; Cloning vectors: plasmids, phagemids; cosmids; YACs, BACs and PACs, expression vectors Library: genomic DNA libraries; cDNA libraries (Isolation of mRNAs, mRNA conversion into double stranded cDNA and Construction of cDNA library); expression libraries; Phage features lgt11; "Chromosome walking"; DNA sequencing; Unidirectional deletions in DNA; "Single stranded conformation polymorphism"; "Southern blot"; "Northern blot"; "Western blot"; Chemical synthesis of oligonucleotides; Determination of the transcription start site; Hybridization "in situ"; "Fingerprinting"; Mutagenesis "in vitro"; radioisotopes; Transgenic animals, "knockout" and knockin '; Tag or reporter gene and selection marker; RNAi: miRNAs and siRNAs and polymorphisms of these RNAs; "Polymerase chain reaction" (PCR): features, Asymmetric PCR, RT-PCR, qPCR, "Ligase Chain Reaction"; "DNA microarrays". Gene Therapy: sense, antisense oligonucleotide with drugs, ribozymes, with vaccines, suicidal with RNAi; Stem cells. RNA sequencing (RNA seq). Chromatin immunoprecipitation (ChIP). Whole Genome Sequencing (WGS). Whole exome sequencing (WES). Next generation sequencing (NGS). MLPA (Multiplex ligation-dependent probe amplification). Genomic editing by CRISPR (clustered regularly interspaced short palindromic repeats)

Applications of Recombinant DNA techniques: PCR and analysis of human genetic diseases; the detection of human-specific sequences; detecting mutations and hereditary diseases; monitoring cancer therapy; detection of viral and bacterial infections; prenatal diagnosis; use of specific oligonucleotides - ASO; molecular evolutionary studies; detection of genetically modified foods; Gene therapy: applications.

Regulation of gene expression in prokaryotes: operon and regulon. Lactose operon: structure and operation; Analysis of mutations and positive control (catabolite repression) of the lactose operon; Arabinose operon; Operon tryptophan and mitigation; Regulation of repair systems; Regulation repressíveis systems; Regulation in translation: Factor Rho; Control of genes of ribosomal proteins; Action ppGpp; Alarmons; Life cycle regulation in l phage.

Regulation of gene expression in eukaryotes: Cell Control Signals: hormones; growth factors; environmental factors. Short term and long term regulation; experimental transcription control. Response levels. Proteins which bind to the promoter; function of enhancers and type of cells involved. Transcription. Heat shock genes; steroid hormone dependent genes; Genes regulated by molecules that bind to receptors; terminal differentiation. Processing and "splicing" alternative: Control the termination of transcription; secondary messengers; Differential processing of pre-mRNA; transcriptional control by overlapping transcriptional units; Transport and stability of mRNA; MRNA translation and protein stability. Control mechanisms: transactivator protein (structure, function); transcriptional control in yeast (expression of GAL genes); Acetylation and methylation; Association with the nuclear matrix. Development in Drosophila: life cycle; development of mature oocyte, embryo (blastoderm syncytial cellular blastoderm, regional specification, homeose and external phenotype). Mutant analysis.

Regulation of the cell cycle: Brief Reference to the different phases of mitosis and meiosis; Cell fusion experiments; external and internal regulators; Control of the transition G1/S (pRb) and intra S (p53); Transition Control G2/M: FPM; Control the transition metaphase / anaphase: bu, mad, Cdc20, APC, separina / separated, condensinas, cohesins, PLK and dawn. Cyclins, CDKs, CKIs; ubiquitination

Oncogenes and anti-oncogenes. Molecular mechanisms of cancer induction: cancer classification, malignant cancers, benign and metastasis. Tumor cells: characteristics. Cause: mutations, cellular changes, biological, occupational, environmental and behavioral, proto-oncogenes change and tumor suppressor genes; chemicals in food, medical factors, social and psychological; virus; epigenetic factors; failure of repair mechanisms, hormonal and reproductive changes, changes in cell proliferation and apoptosis (programmed cell death). Cell cultures: advantages, disadvantages and most common cell lines. Oncogenes: oncogenes cell: Characteristics and classification of oncogenes and their proteins: growth factors; Receptors for growth factors and hormones; intracellular signal transmitters; nuclear transcription factors; Proteins that control the cell cycle. anti-apoptotic proteins. Proteins associated with DNA repair. Anti-oncogenes. viral oncogenes: viral agents of transformation containing DNA; viral agents of transformation containing RNA (Transduction retrovirus; Retroviruses "slow-acting").

 

PROGRAM OF LABORATORY CLASSES

1 - Infection of bacteria by phages.
2 - Kinetics of growth of a bacterial population.
3 - Preparation of competent cells / Research homologies.
4 - Transformation of competent cells with a recombinant DNA.
5 - Plasmid DNA isolation.
6 - Electrophoretic analysis of DNA digested with restriction enzymes.
7 - Preparation of a recombinant plasmid a restriction map and quantification of DNA.
8 - Laboratory exam.

 

Mandatory literature

Videira A ; Engenharia Genética: princípios e aplicações, Lidel, 2001
LeWin Benjamin; Genes VII. ISBN: 0-19-879277-8
Lodish H, Berk A, Kaiser CA, Krieger M, Matthew P, Bretscher SA, Ploegh H & Matsudaira P; (2007). “Molecular Cell Biology”. W. H. Freeman, 6th Ed.
Alberts B, Johnson A, Lewis J & Martin Raff M; (2008). “Molecular Biology of the Cell”. Garland Science, 5thEd.
Bronze-da-Rocha E & Videira A; (2010). “Noções de Genética”, pp 220-259, do livro “Microbiologia”- Lidel Edições Técnicas.

Complementary Bibliography

Sambrook J, Fritsch EF & Maniatis T ; Molecular cloning - a laboratory manual, Cold Spring Harbor Lab. Press, USA, 1989
Arraiano CM & Fialho, A M (; O Mundo do RNA, Lidel, Edições Técnicas, 2007

Teaching methods and learning activities

 

a) Lectures - 3 hours / week; Laboratory classes - 2 hours / week, power-point presentation, the subjects taught in the theory are associated with the practical use of the knowledge imparted. b) Students are encouraged to make bibliographical research and consulting scientific articles related to the subjects taught. c) The laboratory work (2 hours/week) is made individually and according to the established schedule in which the techniques are applied that allow the identification and cloning of genes using the concepts and knowledge acquired in lectures. d) The responsible teacher is available to meet students when requested.

 

 

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Evaluation Type

Distributed evaluation with final exam

Assessment Components

designation	Weight (%)
Exame	80,00
Participação presencial	0,00
Trabalho laboratorial	20,00
Total:	100,00

Amount of time allocated to each course unit

designation	Time (hours)
Estudo autónomo	97,00
Frequência das aulas	65,00
Total:	162,00

Eligibility for exams

Student attendance to the laboratory classes is mandatory. Those students whose attendance is lower than 3/4 of the foreseen classes are considered as without attendance.
- Attendance to the lectures is not compulsory.

Calculation formula of final grade

Examinations or Special Assignments

Students under a special regime, who are not obliged to attend laboratorial classes have to perform the final laboratorial exam before the theretical evaluauation. The approval in this component is mandatory to proceed with the final written evaluation .

Special assessment (TE, DA, ...)

- In accordance with the evaluation rules for FFUP.

Classification improvement

Students have the right to apply for a written test repetition, according to the evaluation standards defined in this curricular unit, for grade improvement, only once, by frequency or by using a resource examination, straightaway subsequent to that in which they passed, and  a new laboratory test is demanded .
- The final subject classification will be the higher of those obtained in the two tests carried out.

Observations

Essential knowledge of Cellular Biology and Bichemistry.
Teaching Language: Portuguese and eventually English.