Introduction to Molecular and Cell Biology Molecular biology seeks to understand the physical and chemical basis of life. and helps us answer the following? What is the molecular basis of disease? What is the molecular basis of evolution? How did life arise on earth? What is the molecular basis of memory? How are different cell types produced from a single embryo? 1
Other Important Questions? What are genes? How do genes store information? How is genetic information expressed? How is this process regulated? How are genes duplicated during cell division? What are mutations? To answer these questions it is necessary to understand the nature of genes and proteins Remembering what you already know 2
DNA Prokaryotic Cell Inner Membrane Cell Wall Outer Membrane Eukaryotic Cell DNA Nucleus Nuclear Membrane Plasma Membrane organelles Prokaryotic Cells: No nuclear membrane No internal compartments Eukaryotic Cells: Nucleus Specialized Organelles 3
Eukaryotic Membranes Permeable to gases and water Active transport requires energy Genetic Information: circular E. coli 4 x 10 6 base pairs of DNA (1 chromosome) max. potential proteins 3 x 10 3 actually 1-21 2 x 10 3 23 pairs Human 2.9 x 10 9 base pairs of DNA (46 chromosomes) cell max. potential proteins 2.4 x 10 6 actually much less (~30-40,000) 100-1000 1000 times the total dry weight of bacteria 4
Human Chromosome pairs Although human beings appear to be much more complex and sophisticated than other organisms, we contain and express very similar genes that are highly evolutionarily conserved The function of some genes is so important, that they are highly conserved in sequence and function 5
Expression of Genes follows an ordered developmental plan HOX Humans and Flies express similar genes but obviously have different pattern of expression (function?) During evolution, it is easier to adapt an existing gene than to create it from scratch Biological molecules are interdependent DNA Central Dogma RNA Protein You need protein to make DNA/RNA and you need nucleic acids to make proteins 6
Prevalent view in early 1900 s s was that genetic information was contained within proteins Why? Proteins are more complex than nucleic acids (20 amino acids vs 4 different nucleotides) Nucleic acids, DNA, was believed to play structural role in cell DNA is the Genetic Material n Early 1940 s DNA was finally implicated as the genetic material n Pure DNA extracted from one bacterial strain could provide genetic information to another bacteria by a process known as transformation n Makes sense since DNA is very stable, it is present in two copies in eukaryotes (diploid), exist as double-stranded molecule, and most importantly can be faithfully copied 7
Genes Defined as a unit of DNA that encodes the information for the synthesis of a protein Prokaryotes contain less genetic information (genes) than eukaryotes Genes are copied (transcribed) into messenger RNA and it is this message that is translated into protein In prokaryotes, genes involved in the same pathway are commonly linked closed together In most eukaryotes, each gene is generally independently copied or transcribed into a single RNA that is then translated into a single protein Three genes in two chromosomes 8
Most genes in mammals and plants contain introns that are removed during RNA processing Introns spliced out (deleted) mature message Exons encode the information for protein synthesis Mutations within genes can cause disease or death Mutation in an exon can cause the production of an abnormal or truncated (shorter) protein Mutation in an intron can cause no effect or can alter or destroy the normal processing of the mrna Mutation in regulatory regions can cause the gene to not be expressed at all or over-expressed 9
Alternative Splicing can produce different or altered proteins from the same gene Fig.9-2 Even though humans encode for ~30,000 proteins (based on gene count), the # of different proteins is higher due to added complexity 10
In eukaryotes, each gene is independently copied and generally encodes information for a specific product (protein) Eukaryotic mrna is a product of several modifications which include removal of introns All DNA is arranged in a structure called a double-helix that is composed of two identical strands which adds to the chemical stability of this molecule Watson & Crick DNA Double Helix (1953) 11
Linkage of Nucleic Acids Base 1 Base 2 5 O 3 Sugar OH HO P O Sugar O - -H2O 5 Base 1 Sugar O O P O - O Base 2 Sugar 3 O - O - P O O CH 2 O Base 1 H O - H O P O H H O H Base 2 CH 2 O H H H 3 OH H H 12
A C T A G P OH 5 ACTAG 3 UGAUC T only in DNA And U only in RNA DNA Helix Held by many H bonds and Hydrophobic Interactions Bases stack on inside Sugar Phosphate backbone is on the outside 13
To maintain the geometry of this structure small bases, pyrimidines, (C or T) must pair with larger bases, purines, (A or G) 14
Thymidine (T) CH 3 C O H N H C N H HC C O N C (Deoxyribose) A-T T Base Pair N HC Adenine (A) C N C C N CH N C (Deoxyribose) Base pair complementarity due to size, shape, and chemical composition of bases C-G G Base Pair Cytosine (C) H N H HC C N HC C O N C (Deoxyribose) H H N H Guanine (G) N C O C C N CH C N N C (Deoxyribose) 15
Genes are arranged in Chromosomes in specialized structures Widely separated from each other Humans and other higher organisms have more junk DNA 16
DNA packaging within cells The E. coli bacterial genome is approximately 1mm long which is about 1000 x the size (vol( vol) ) of a single bacteria DNA needs to be highly compacted Eukaryotic DNA n Can be up to hundred thousand times longer than the cell that contains it n Found in highly compacted units called chromosomes n DNA is wound around special proteins 17
Eukaryotic genes are arranged in chromosomes gene 1 gene 2 gene 3 Longest human chromosomes 2-33 x 10 8 base pairs ~10 cm long gene 4 Contents of Chromosomes Chromosomes contain different types of sequences such as: n Single copy genes, gene families, defective genes n Non-coding DNA and repetitive sequences (can compose a significant part of genome) n Viral DNA and other transposable elements (few) 18
DNA cloning Cloning comes from the greek word klon which means twig Plants can be cloned by taking a cutting and replanting them- they should turn out identical to original plant. The first cloning experiment was performed in 1973 (only 37 yrs ago) by Cohen and Boyer They used bacterial plasmids which are small circular replicating fragments of DNA They also used enzymes that cut DNA into specific fragments. These enzymes are called restriction endonucleases (enzymes that cleave nucleic acids) Plasmids are selfish pieces of DNA that are circular and replicate inside of bacterial host Origin of replication Ampicillin resistance gene 19
When plasmids replicate they make two identical copies Fig. 7.2 Restriction Enzyme Cleaves DNA at specific sites They are called sticky ends because they have a tendency of pairing ing or re-pairing with each other (or other foreign fragments) 20
Fragment of foreign DNA can be readily introduced into plasmids Mix plasmid with E. coli bacteria and perform transformation Heat Shock 21
Restriction Enzymes Restriction enzymes (RE) are the bacteria s s defense against viruses These enzymes restrict the host range of the viruses There are more than 100 different restriction enzymes from a large number of different bacteria Biotech companies have been making millions of dollars selling specific enzymes to researchers 22