Peter John M.Phil, PhD Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences & Technology (NUST)

DNA, Genes & Genome Gene: A sequence of DNA that code for an RNA, which in turn code for protein DNA is the genetic material of all the living organisms In some viruses the genetic material is RNA

Human Genome Genome: complete set of instructions for making an organism is called its genome This information/sequence is used to construct the proteins, which are the structural part of the organism or used in metabolic reactions necessary for life Human genes 30,000. 10% of the genome is known to be coding

Amino Acids & Proteins 3 bases code for 1 amino acid Twenty different kinds of amino acids are usually found in proteins Humans can synthesize at least 100,000 different kinds. Proteins instructions from the genes are transmitted indirectly through mRNA mRNA is moved from the nucleus to the cellular cytoplasm, where it serves as the template for protein synthesis.

Organization of the Human Genome Nuclear genome (3300 Mb , ~80,000 genes) Mitochondrial Genome (16.6 kb, 37 genes) Nuclear Genome (25% Genes & genes related sequences, 75% extragenic DNA) Extragenic DNA (Repetitive Sequences) Genes & Genes Related Sequences (10% coding DNA, 90% non coding DNA) Non Coding DNA (Pseudogenes, Gene Fragments, Untranslated Sequences)

Mitochondrial Genome Size 16.6 kb, 37 genes 2, rRNA genes, 22, tRNA, 13 polypeptide noncoding genes

Organization of the Human Genome

Central Dogma Central dogma states that information in nucleic acid can be perpetuated or transferred but the transfer of information into protein is irreversible

Central Dogma

Mutation A point mutation changes a single base pair Point mutations can be caused by the chemical conversion of one base into another or by mistakes that occur during replication. Transition Mutation: mutation in which a purine/pyrimidine base pair is replaced with a base pair in the same purine/pyrimidine relationship replaces a G-C base pair with an A-T base pair or vice-versa. A transversion replaces a purine with a pyrimidine, such as changing A-T to T-A. Insertions are the most common type of and result from the movement of transposable elements

Loss/Gain of function Recessive mutations are due to loss of function by the protein product Dominant mutations result from a gain of function Silent mutation have no effect on the protein function leaky mutation: Enough active product is made to fulfill its function, even though the activity is quantitatively reduced or qualitatively different from the wild type.

Interrupted genes and Alleles An interrupted gene consists of exons and introns The exons are the sequences represented in the mature RNA. By definition, a gene starts and ends with exons, corresponding to the 5' and 3' ends of the RNA. The introns are the intervening sequences that are removed when the primary transcript is processed to give the mature RNA.

Interrupted genes and Alleles A gene may exist in alternative forms which are called alleles Genetic locus: Each gene reside on a particular location on a chromosome which is called as genetic locus

Multiple Alleles Different variants of the same gene are called multiple alleles, Each of these mutant alleles must therefore represent a different mutation of the gene, which does not eliminate its function entirely, but leaves a residual activity that produces a characteristic phenotype. When multiple alleles exist, an animal may be a heterozygote that carries two different mutant alleles.

Cis & Trans-acting element Cis Acting Elements DNA sequences in the vicinity of the structural portion of a gene that are required for gene expression Trans Acting Elements factors, usually considered to be proteins, that bind to the cis-acting sequences to control gene expression

Cis Acting Elements Promoters (200 bp up stream, serve to initiate transcritpion) Enhancer (200-300bp up stream or integral components of promoter, recognize ubiquitous transcription factor/tissue specific transcription factor) Silencer elements (reduce transcription level, reported at various position within promoter)

Cis Acting Elements Boundary Elements sequences that define the boundaries of coordinately regulated chromatin domains in chromosomes. Response Elements (Give response to certain stimuli, upstream of promoter)

Trans Acting Elements subunits of RNA polymerase Transcription Factors Regulatory Proteins

Transcription Factors Recognize target sequences in DNA Interact with other transcription factors TF contain conserved motifs that permits DNA binding Activation Domain Binding Domain

Cis & Trans-acting element

Protein Expression Several processes are required to express the protein product of a gene A gene is expressed by transcription in to mRNA then by translation of the mRNA into protein In eukaryotes a gene contain internal region/introns, which are removed by splicing mechanism to give mRNA

Control of Gene Expression At Transcriptional Level (Cis, Trans acting elements) At Post Transcriptional Level (at RNA level, RNA splicing, mRNA transport, translation, mRNA stability, Protein processing, Protein stability) Epigenetic Mechanism (factor which are not directly attributed to DNA sequences)

Genetic Code Genetic code: The relationship between a sequence of DNA and the sequence of the corresponding protein. The genetic code is read in groups of three nucleotides, each group representing one amino acid. Codon: Each trinucleotide sequence is called a Codon.

Genetic Code The triplets are nonoverlapping and are read from a fixed starting point. Mutations that insert or delete individual bases cause a shift in the triplet sets after the site of mutation. Combinations of mutations that together insert or delete 3 bases (or multiples of three) insert or delete amino acids but do not change the reading of the triplets beyond the last site of mutation.

Genetic Code Three types of RNAs: mRNA: messenger RNA Contains 3 bases ( codon) rRNA: ribosomal RNA Comprises the 70 S ribosome tRNA: transfer RNA Transfers amino acids to ribosomes for protein synthesis Contains the anticodon (3 base sequence that is complimentary to codon on mRNA)

Genetic Codes The seq of coding strand DNA start in 5’---3’ direction It consist of NT triplet called codons, which represent one AA Four NT (A, T, G, C) makes 64 possible codons 61 codons are involve in protein synthesis, 3 are terminating codons

Condon & Amino Acids Sixty one of the sixty four possible triplets code for twenty amino acid Three codons don’t represent amino acids and cause termination Most amino acids are represented by more than one codon

Condon & Amino Acids Multiple codons for an amino acid are usually related Related amino acids often have related codons, minimizing the effect of mutation Identical set of codon assignments is used in bacteria and eukaryotic cytoplasm By this mRNA from one species can translated invitro/vivo by protein synthetic apparatus of other species

Genetic Codes

Codon & Anticodon The meaning of the codon is determined by tRNA that correspond to it The assignment of the AAs to the codon is not random, but shows a relationship b/w codon & anti codon

Related Codon represent related AAs There are more codons than AAs, as a result all AAs are represented by more than 1 codon Only 2 AAs Met & Trp have single set of codon & are called “ synonymous” Codon representing the same AA have same seq only with the diff at position at 3, this is called “ third base degeneracy”

Ribosome This codon/anticodon interaction is stabilized by the environment of the ribosomal “A’ site. Then ribosome control the environment & pairing occur b/w first 2 bases & additional reactions are permitted at third base.

Third base degeneracy The third base degeneracy reduce the effect of mutation b/c no change in AA occur The three codons, UAA, UAG, UGA are stop/terminating codons mark the end of gene

Codons in other species The same set of genetic code is used in bacteria and eukaryotic cytoplasm Thus codon use in one specie have the same meanings for the ribosome of the other specie Any change in the genetic code result in the disruption of the protein

Meanings of Codons in other species Sometimes change in the meanings of the codons are different in different species, e.g. In mycoplasma: UGA (stop)------ code for Trp In certain ciliates: UAA & UAG (stop)------ code for Glutamine

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