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Genetic Engineering.pptx

  1. 3/29/2023 OSAMAH S. ALRAWOUAB 1
  2. 3/29/2023 OSAMAH S. ALRAWOUAB 2
  3. 3/29/2023 OSAMAH S. ALRAWOUAB 3 Genome  Genome is the complete set of genetic information of a cell or an organism in particular, the complete sequence of DNA/RNA that carries this information.  In diploid organisms, it refers to the haploid set of chromosomes present in a cell.  Depending on its localization, genome may be nuclear or organellar.  Organellar genomes are again of two types: mitochondrial and chloroplast genome.  Genome size of organisms differs significantly between different species. The size of the genome governs the size and complexity of an organism. However, many small sized organisms, in fact have bigger genomes than their larger counterparts.
  4. 3/29/2023 OSAMAH S. ALRAWOUAB 4 Genome
  5. 3/29/2023 OSAMAH S. ALRAWOUAB 5 Genome The genome contains all the genes present in the nucleus of a cell. Gene varies in size from a few hundred DNA/RNA bases to more than few thousand bases.  The haploid set of chromosome contains the total genome of the organism.  The bacterium Mycoplasma genitalium has a small genome size of 0.58Mb and the plant Triticum aestivim has a large genome size of 16000Mb. The genome size in human is 3200Mb.
  6. 3/29/2023 OSAMAH S. ALRAWOUAB 6 Genome
  7. 3/29/2023 OSAMAH S. ALRAWOUAB 7 Role of genes within cells Genes contain the instructions for each cell to make proteins and RNAs. Genes are made up of DNA fragments. Within the cell the DNA performs two tasks:  Act as information repository including instructions in making the component molecules of the cells.  Pass on the information to the next generation.  The mere presence of DNA does not implicate a cell to be alive and functional. Mammalian red blood cells (RBCs) discard nucleus during developmental process and thus lacks DNA in mature state.
  8. 3/29/2023 OSAMAH S. ALRAWOUAB 8 Role of genes within cells Genes are transcribed to RNA which are processed to various forms like mRNA, tRNA, rRNA etc. mRNA are translated to proteins depending on the regulatory signals. tRNA and rRNA serve as the components of translational machinery. New functions of RNA are also being discovered like regulatory (miRNA, siRNA etc) and catalytic (ribozymes) functions.
  9. 3/29/2023 OSAMAH S. ALRAWOUAB 9 Gene expression
  10. 3/29/2023 OSAMAH S. ALRAWOUAB 10 Gene expression 1- Access to gene: Genes are inaccessible as they are buried deep within the highly packaged chromosomes. The initial step involves a preparative process that opens the chromatin structure and positions of the nucleosome in the region of genome containing active genes. Events involved in gene expression
  11. 3/29/2023 OSAMAH S. ALRAWOUAB 11 Gene expression 2-Formation of transcription initiation complex: involves the assembly of a set of proteins into a complex that copy DNA into RNA. This is a highly regulated process as the transcription initiation complex must be constructed at the precise position in the genome, adjacent to active genes to form a RNA copy. Events involved in gene expression
  12. 3/29/2023 OSAMAH S. ALRAWOUAB 12 Gene expression Events involved in gene expression
  13. 3/29/2023 OSAMAH S. ALRAWOUAB 13 Gene expression 3- RNA Synthesis involves the transcription of a gene into RNA molecule and it occurs in the nucleus. Events involved in gene expression
  14. 3/29/2023 OSAMAH S. ALRAWOUAB 14 Gene expression 4- RNA Processing comprises of post transcriptional modification/alterations of the RNA molecule and its chemical structure required for the RNA to be translated into protein or non-coding RNA (rRNA, tRNA, miRNA). RNA splicing (deletion of introns and combination of exons), 5’ capping, polyadenylation etc are commonly occurred RNA processing steps in eukaryotes. However, prokaryotic organisms do not have a well developed RNA processing mechinary. Events involved in gene expression
  15. 3/29/2023 OSAMAH S. ALRAWOUAB 15 Gene expression 5- Degradation of RNA is the controlled turnover of RNA molecules and should not be viewed simply as a mean of getting rid of unwanted RNAs. It determines the makeup of the transcriptome and is considered as an important step in genome expression. Different ribonucleases (RNases) plays the prime role in this process and multiple cofactors like small RNA (siRNA, miRNA etc), molecular cheparons (Lsm1-7, Lsm2-8, Hfq etc) regulate this process. Events involved in gene expression
  16. 3/29/2023 OSAMAH S. ALRAWOUAB 16 Gene expression 6- Protein synthesis is initiated after the assembly of the translation initiation complex near the 5’ termini of a mRNA molecule. It involves translation of RNA molecules into proteins. Events involved in gene expression
  17. 3/29/2023 OSAMAH S. ALRAWOUAB 17 Gene expression 7- Protein folding and protein processing may occur together after protein synthesis. Post translation events like folding involve the protein attaining its correct three dimensional configuration. Processing (phosphorylation, glycosylation, carboxylation etc.) involves the modification of the protein by addition of chemical groups and removal of one or more functional units of the protein. Events involved in gene expression
  18. 3/29/2023 OSAMAH S. ALRAWOUAB 18 Gene expression 1- Constitutive expression: Housekeeping genes are essential and necessary for sustaining life, and are therefore continuously expressed. gapdh (glyceraldehydes 3 phosphate dehydrogenase), sdha (succinate dehydrogenase) etc are human housekeeping genes which are expressed throughout the development. Types of gene expression
  19. 3/29/2023 OSAMAH S. ALRAWOUAB 19 Gene expression 2- Induction and repression: The expression levels of some genes fluctuate in response to external signals. Also, under a certain situation, some genes show higher expression level, while others show lower expression levels. The former is called induced expression and the latter is called repressed expression. Types of gene expression
  20. 3/29/2023 OSAMAH S. ALRAWOUAB 20
  21. 3/29/2023 OSAMAH S. ALRAWOUAB 21 A. Codons  Codons are presented in the messenger RNA (mRNA) language of adenine (A), guanine (G), cytosine (C), and uracil (U).  Their nucleotide sequences are always written from the 5′ end to the 3′ end.  The four nucleotide bases are used to produce the three-base codons. There are, therefore, 64 different combinations of bases
  22. 3/29/2023 OSAMAH S. ALRAWOUAB 22 A. Codons How to translate a codon
  23. 3/29/2023 OSAMAH S. ALRAWOUAB 23 A. Codons  This table (or “dictionary”) can be used to translate any codon sequence and, thus, to determine which amino acids are coded for by an mRNA sequence.
  24. 3/29/2023 OSAMAH S. ALRAWOUAB 24 A. Codons Termination (“stop” or “nonsense”) codons Three of the codons, UAG, UGA, and UAA, do not code for amino acids but rather are termination codons. When one of these codons appears in an mRNA sequence, it signals that the synthesis of the protein coded for by that mRNA is complete.
  25. 3/29/2023 OSAMAH S. ALRAWOUAB 25 A. Codons Termination (“stop” or “nonsense”) codons
  26. 3/29/2023 OSAMAH S. ALRAWOUAB 26 A. Codons Characteristics of the genetic code 1. Specificity: The genetic code is specific (unambiguous), that is, a particular codon always codes for the same amino acid. 2. Universality: The genetic code is virtually universal, that is, the specificity of the genetic code has been conserved from very early stages of evolution. (Note: An exception occurs in mitochondria, in which a few codons have meanings different.
  27. 3/29/2023 OSAMAH S. ALRAWOUAB 27 A. Codons Characteristics of the genetic code 3. Degeneracy: The genetic code is degenerate (sometimes called redundant). Although each codon corresponds to a single amino acid, a given amino acid may have more than one triplet coding for it. For example, arginine is specified by six different codons 4. Nonoverlapping and commaless The genetic code is nonoverlapping and commaless, that is, the code is read from a fixed starting point as a continuous sequence of bases, taken three at a time. For example, ABCDEFGHIJKL is read as ABC/DEF/GHI/JKL without any “punctuation” between the codons.
  28. 3/29/2023 OSAMAH S. ALRAWOUAB 28 A. Codons Consequences of altering the nucleotide sequence Changing a single nucleotide base on the mRNA chain (a “point mutation”) can lead to any one of three results: 1. Silent mutation: The codon containing the changed base may code for the same amino acid. For example, if the serine codon UCA is given a different third base “U” to become UCU, it still codes for serine. This is termed a “silent” mutation.
  29. 3/29/2023 OSAMAH S. ALRAWOUAB 29 A. Codons Consequences of altering the nucleotide sequence Changing a single nucleotide base on the mRNA chain (a “point mutation”) can lead to any one of three results: 2. Missense mutation: The codon containing the changed base may code for a different amino acid. For example, if the serine codon UCA is given a different first base “C” to become CCA, it will code for a different amino acid, in this case, proline. The substitution of an incorrect amino acid is called a “missense” mutation.
  30. 3/29/2023 OSAMAH S. ALRAWOUAB 30 A. Codons Consequences of altering the nucleotide sequence Changing a single nucleotide base on the mRNA chain (a “point mutation”) can lead to any one of three results: 3. Nonsense mutation: The codon containing the changed base may become a termination codon. For example, if the serine codon UCA is given a different second base “A” to become UAA, the new codon causes termination of translation at that point and the production of a shortened (truncated) protein. The creation of a termination codon at an inappropriate place is called a “nonsense” mutation.
  31. 3/29/2023 OSAMAH S. ALRAWOUAB 31 A. Codons Other mutations  Occasionally, a sequence of three bases that is repeated in tandem will become amplified in number so that too many copies of the triplet occur.  If this occurs within the coding region of a gene, the protein will contain many extra copies of one amino acid. For example, amplification of the CAG codon leads to the insertion of many extra glutamine residues in the Huntington protein, causing the neurodegenerative disorder, Huntington disease. a. Trinucleotide repeat expansion:
  32. 3/29/2023 OSAMAH S. ALRAWOUAB 32 A. Codons Other mutations  The additional glutamines result in unstable proteins that cause the accumulation of protein aggregates. If the trinucleotide repeat expansion occurs in the untranslated portion of a gene, the result can be a decrease in the amount of protein produced as seen, for example, in fragile X syndrome and myotonic dystrophy. a. Trinucleotide repeat expansion:
  33. 3/29/2023 OSAMAH S. ALRAWOUAB 33 A. Codons Other mutations  Mutations at splice sites can alter the way in which introns are removed from the pre-mRNA molecules,  producing aberrant proteins. b. Splice site mutations:
  34. 3/29/2023 OSAMAH S. ALRAWOUAB 34 A. Codons Other mutations If one or two nucleotides are either deleted from or added to the coding region of a message sequence, a frame-shift mutation occurs and the reading frame is altered. This can result in a product with a radically different amino acid sequence or a truncated product due to the creation of a termination codon. c. Frame-shift mutations:
  35. 3/29/2023 OSAMAH S. ALRAWOUAB 35 A. Codons Other mutations If three nucleotides are added, a new amino acid is added to the peptide, or if three nucleotides are deleted, an amino acid is lost. In these instances, the reading frame is not affected. Loss of three nucleotides maintains the reading frame but can result in serious pathology. c. Frame-shift mutations:
  36. 3/29/2023 OSAMAH S. ALRAWOUAB 36 A. Codons Other mutations For example, cystic fibrosis (CF), a hereditary disease that primarily affects the pulmonary and digestive systems, is most commonly caused by a deletion of three nucleotides from the coding region of a gene, resulting in the loss of phenylalanine at the 508th position (ΔF508) in the protein encoded by that gene. This ΔF508 mutation prevents normal folding of the CF transmembrane conductance regulator (CFTR) protein c. Frame-shift mutations: