Lecture 2 Reading PDF - BIOM-2131 Introductory Molecular Biology - W2025

BIOM-2131 Introductory Molecular Biology LECTURE 2 From DNA to GENES LECTURE OVERVIEW ▪ 2.1 DNA ▪ 2.2 Chromosomes ▪ 2.3 Genes and Genomes ▪ 2.4 DNA sequencing ▪ 2.5 SNPs ...

BIOM-2131 Introductory Molecular Biology LECTURE 2 From DNA to GENES LECTURE OVERVIEW ▪ 2.1 DNA ▪ 2.2 Chromosomes ▪ 2.3 Genes and Genomes ▪ 2.4 DNA sequencing ▪ 2.5 SNPs  Deoxyribonucleic acid DNA is a long polymer composed of four types of nucleotide subunits Nitrogen-containing bases building blocks of DNA the nucleotide subunits within a DNA 5’ end strand are 5’ held 4’ 1’ together by 3’ 2’ 5’ DNA strand 4’ 1’ phosphodiester 3’ 2’ bonds Alberts, et. al, Essential Cell Biology, 6th edition, Figure 5-2 (partial), p. 181 https://opentextbc.ca/biology/chapter/9-1-the-structure-of-dna/ www.scienceabc.com/pure-sciences/what-is-a-phosphodiester-bond.html 3’ end  Deoxyribonucleic acid the nucleotides are linked together covalently by phosphodiester bonds that connect the 3’-hydroxyl group (-OH) of one sugar and the 5’ phosphate group attached to the next gives rise to polarity of the resulting DNA strand double-strand DNA DNA double helix 5’ 3’ 3’ 5’ arrows indicate polarity of DNA strand antiparallel Alberts, et. al, Essential Cell Biology, 6th edition, Figure 5-2 (partial), p. 181 Voet, D., et al, Fundamentals of Biochemistry, Figures 3-5, 3-6, p. 47, 48  Deoxyribonucleic acid bases lie perpendicular to the axis of the helix double helix held together by hydrogen bonding between bases on different strands complementary base pairing A T C G the bases can pair this way only if the two chains are antiparallel (oriented in opposite direction) Alberts, et. al, Essential Cell Biology, 6th edition, Figure 5-4, p. 183  Eukaryotic DNA - packaged into chromosome nucleosomes fit inside nucleus are basic units of eukaryotic chromosome structure interphase DNA + chromosomes specialized after duplication proteins – histones mitotic chromosomes accurate apportioning Keating ST, et al, Circ Res. 2016. 118(11):1706-22 www.nature.com/scitable/topicpage/chromosomes-14121320/ between 2 daughter cells  Eukaryotic DNA is packed into chromatin which further packs into chromosomes each human cell contains about 2 meters of DNA the cell nucleus is about 5-8 m in diameter https://dnalc.cshl.edu/resources/3d/08-how-dna-is-packaged-advanced.html  Is the entire interphase chromatin packed the same way? the interphase chromatin is not uniformly packed the detailed structure of interphase chromatin organization differ from one cell type to the next most cell types express only ~ ½ of the genes they contain and many of these are at low level actively expressed contain silent genes genes more extended more condensed regions regions www.researchgate.net/figure/Chromatin-folding-The-first-level-of-compaction-is-the-association-of-DNA-with_fig1_300903837  Interphase chromatin contains both, heterochromatin the most highly condensed form makes up about 40% of typical chromosome about ½ remains permanently condensed e.g. regions of centromere and telomeres other ½ contains genes whose activity has been silenced euchromatin less condensed state than heterochromatin about 60% of typical chromosome Alberts, et. al, Essential Cell Biology, 6th edition, Figure 5-29, p. 198  Eukaryotic DNA is packed into multiple chromosomes the DNA in a human nucleus is parceled out into 23 or 24 different types of chromosomes human chromosomes come in different sizes can be distinguish from one another using a variety of molecular and microscopic techniques each chromosome can be “painted” a different colour to allow its unambiguous identification FISH karyotype SKY Alberts, et. al, Essential Cell Biology, 6th edition, Figure 5-8 (partial), p. 185  Chromosomes organize and carry genetic information most important function of a chromosome carry genes functional units of heredity a gene can be defined as a segment of DNA that contains the instructions for making a particular protein or RNA molecule Alberts, et. al, Essential Cell Biology, 6th edition, Figure 5-7, p. 184  From genes to genomes the total genetic information carried by a complete set of the chromosomes present in a cell or organism constitutes its genome complete genome sequences many thousands organisms from E. coli to humans Organisms vary enormously in the size of their genome Alberts, et. al, Essential Cell Biology, 6th edition, Table 1-2, p. 38  Genes in… bacteria and some many eukaryotes single cell eukaryotes, including S. cerevisiae closely packed genes – compact genomes genes include an excess of interspersed, human genome is 200x noncoding larger than S. cerevisiae DNA Alberts, et. al, Essential Cell Biology, 6th edition, Figure 5-10, p. 186 Alberts, et. al, Essential Cell Biology, 6th edition, Figure 5-11, p. 187  How DNA is apportioned over chromosomes … …. differs from one species to another humans have 46 chromosomes, including both maternal and paternal sets a species of small deer – only 7 some carp species – more than 100  How DNA is apportioned over chromosomes closely related species with similar genome sizes can have very different chromosomal numbers and sizes muntjac Alberts, et. al, Essential Cell Biology, 6th edition, Figure 5-12, p. 188  From genes to genomes gene number is roughly correlated with species complexity there is no simple relationship between gene number, chromosome number and total genome size  Human genome 1st draft: 1999 complete sequence: 2021 complete genetic landscape: how many genes we have? what those genes look like? how they are distributed across the genome?  Bulk of human genome is made of …. repetitive nucleotide sequences and other non-protein-coding DNA LINEs: long interspersed nuclear elements (L1) SINEs: short interspersed nuclear elements (Alu) most of these mobile simple repeats genetic elements are segment duplications remnants – no longer most highly repetitive DNA capable of transposition heterochromatin – 10% Alberts, et. al, Essential Cell Biology, 6th edition, Figure 9-32 (partial), p. 333  About half of human genome consists of …. unique sequences that are not part of any introns or exons: regulatory DNA sequences sequences that code for functional RNA sequences whose functions are not known yet less than 1 % of human genome represents protein-coding exons Alberts, et. al, Essential Cell Biology, 6th edition, Figure 9-32 (partial), p. 333  Human genome number of protein-coding genes in the human genome may be unexpectedly small only about 1,300 nucleotide pairs are needed to encode an average size protein of about 430 amino acids average length of human gene is 26,000 nucleotide pairs most of this DNA noncoding introns regulatory DNA sequences typically interspersed along tens of thousands nt pairs  Genes are sparsely distributed in human genome ~ 50,000 nt pairs compared to many other eukaryotic genomes, the human genome is much less densely packed Alberts, et. al, Essential Cell Biology, 6th edition, Figure 9-33, p.334  Human genome exons comprise less than 1% comparative studies indicate that about 4.5% of the human genome is highly conserved when compared with other mammalian genomes additional 5% of the genome shows reduced variation in the human population  Differences in gene regulation… … help explain how animals with similar genomes can be so different regulatory DNA sequences associated with each gene dictate each organisms developmental program Alberts, et. al, Essential Cell Biology, 6th edition, Figure 9-34, p. 335  DNA Sequencing Two methods originally developed Maxam-Gilbert method chemical cleavage of DNA at specific nt Sanger method enzymatic extension of DNA strand to a defined terminating base both methods can determine sequence of 500- 700 bp per reaction and have 99.9% accuracy  DNA sequencing: Sanger method Template DNA is denatured and mixed with radiolabeled oligonucleotide primer, dNTPs, and DNA polymerase Sample is spit into four aliquots; each aliquot receives a different dideoxyribonucleotide (ddNTP) During DNA synthesis, ddNTPs are incorporated into DNA like dNTPs, but lack 3’OH group so cannot be extended  DNA sequencing: Sanger method Hartwell, L.H, et al, Genetics: From Genes to Genomes, Figure 9.13 (partial), Page 314  Automated DNA sequencing single stranded DNA template + sequencing primers incubated in the presence of small amount of four fluorescently labelled chain- terminating ddNTPs are amplified by DNA polymerase electrophoresis in capillary tube fluorescence of each fragment identifies its 3-terminal nucleotide Voet, D., et al, Fundamentals of Biochemistry, Figures 3-19, 3-20, p. 58 & 59  Central dogma of molecular biology the flow of genetic information in cells is from DNA to mRNA to protein transcription a process in which cells synthesise mRNA using DNA as a template translation a process in which cells use the information in mRNA to make protein Voet, D., et al, Fundamentals of Biochemistry, 4th edition, Figure 3-12, p. 52  How cell read their genomes? flow of genetic information from DNA to mRNA to protein https://dnalc.cshl.edu/resources/3d/central-dogma.html  Human gene → protein simplified schematics www.genome.gov/genetics-glossary/Exon#:~:text=An%20exon%20is%20a%20region,consist%20of%20exons%20and%20introns.  From mRNA to cDNA (in a test tube) mRNA (cells, tissues) mRNA → cDNA (lab) purify mRNA (lab) oligo(dT) primer www.genome.gov/genetics- recombinant RT glossary/Exon#:~:text=An%20exon%20is%20a%20region,consist%20of%20exons%20and%20int rons. schematics from www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553/  Identifying protein-coding genes can protein coding sequence be identified in genomic sequence? bacteria and simple eukaryotes (yeast) Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-19, p. 249  Identifying protein-coding genes specific start signal for translation (in cells) AUG = methionine # of possible reading frames? Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-30, p. 256  Identifying good candidate for protein-coding genes ORFs - open reading frames sequences  10,000 codons that lack stop codon a random sequence will have by chance a stop codon every 20 nucleotides continuous nucleotide sequence that encode more than 100 amino acids Alberts, et. al, Essential Cell Biology, 6th edition, Figure 9-35, p. 336  Single-nucleotide polymorphisms (SNPs) single-base changes that are present in at least 1% of the populations two human genomes chosen at random from the world’s population will differ by ~2.5 x 106 SNPs that are scattered throughout the genome Alberts, et. al, Essential Cell Biology, 6th edition, Figure 9-38, p. 339  An Example of SNP and changes in regulation of a gene ability of some adults to digest milk enzyme lactase digests lactose (main sugar in milk) ancestors: express during infancy domestication of cattle ~ 10,000 years ago random mutations – express during adulthood Alberts, et. al, Essential Cell Biology, 6th edition, Figure 9-6, p. 313  Lecture 2 Reading Chapter 5: DNA and Chromosomes pages 179-192; 197-199 Chapter 9: Examining the Human Genome pages 331-339  BIOM-2131 Labs start on Monday, January 13, 2025 must attend lab section you registered for on UWinsite student!!! need electronics!!!  BIOM-2131 Labs GA/TA support until end of regularly scheduled lab time labs due on D2L end of lab time deadline on D2L unavailable check UWinsite for location of your lab section in Dillon Hall Coming up: Lecture 3 Reading Chapter 6: DNA Replication and Repair pages 179-225