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DNA from the Beginning- Genetic Organization and Control Adapted from http://www.dnaftb.org Copyright, Cold Spring Harbor Laboratory

Introduction: Progress in understanding how DNA, RNA, proteins and other molecules interact leads to more discoveries. The use of tools; enzymes, cell free systems, purification methods, model organisms, structure determination, microscopes, computers, etc., allows more and more discovery,

Before class: Look at the following concepts. Write down any words you don’t know. If you don’t understand the concept, please read the following notes carefully. Be prepared to ask questions in class. After we have discussed your questions, there will be a short test of these concepts in class. Concept 29 - DNA is packaged in a chromosome http://www.dnaftb.org/29 : http://www.dnaftb.org/29/animation.html Concept 30 – Higher cells incorporate an ancient chromosome http://www.dnaftb.org/30 : http://www.dnaftb.org/30/animation.html Concept 31 - Most DNA does not encode protein http://www.dnaftb.org/31 : http://www.dnaftb.org/31/animation.html Concept 32 - Some DNA can jump http://www.dnaftb.org/32 : http://www.dnaftb.org/32/animation.html Concept 33 - Genes can be turned on and off http://www.dnaftb.org/33 : http://www.dnaftb.org/33/animation.html

Concept 29 - DNA is packaged in a chromosome. Living things have a set of chromosomes in the nucleus of each cell. The chromosomes are made of DNA and proteins. Each chromosome is a package for one very long, continuous strand of the DNA. Structural proteins, including histones, combine with DNA to make a compact chromosome. The DNA strand is wound around histone cores, which are looped and fixed to specific regions of the chromosome.

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Kornberg and Klug

class of proteins - histones and how they interact with DNA.

five different kinds of histones.

histones bind to DNA to form the chromatin in the nucleus of higher cells.

in non-dividing cells, the chromatin is dispersed throughout the nucleus.

prophase of cell division, the chromatin condenses into the visible structures.

a cell in metaphase; the chromosomes are lined up in the middle of the cell.

X-ray diffraction studies show histones are important to give structure for the DNA helix.

Wilkins and Luzzati - 1964 - chromatin has a repeating pattern with intervals of about 100

angstroms (1Å = 10 -10m).

Klug - similar X-ray diffraction patterns in chromatin.

repeat suggested that histones play a role in "packaging" DNA.

Hewish, and Burgoyne in 1973 - used DNA nuclease to digest chromatin.

electrophoresed the digested chromatin material

a regular pattern of bands on the gel.

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multiples of the smallest size fragment, about 200 base pairs (bp).

repeated bands corresponded to 200 bp, 400 bp, 600 bp, 800 bp, and so on.

concluded - the histones are distributed evenly on the DNA and, where they bind, protect the DNA from nuclease digestion.

DNA without histones, digested with nuclease produces a smear of thousands of different-sized fragments.

the X-ray diffraction patterns and the nuclease experiments -> chromatin is DNA wrapped around the histone cores.

The 200 bp repeat observed after nuclease digestion corresponds to 200 bp of DNA wrapped around each histone core.

The 100 Å measurement from X-ray diffraction patterns is the width of the histone core and the DNA.

individually purified the histones from the DNA. - H2A and H2B stick together, H3 and H4 stick together.

mix the H2A/H2B complex with the H3/H4 complex, and then added naked DNA, -> the same X-ray pattern as for chromatin.

each histone core has eight proteins -- two copies each of the H2A/H2B and H3/H4 complexes.

histone core with wrapped DNA is called a nucleosome.

electron micrograph of chromatin.

The "string" is called the 10-nm fiber. The "beads" are the nucleosomes.

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H1 is not part of the histone core but binds between nucleosomes to give even more structure to chromatin.

H1 sits just outside of each nucleosome and interacts with the H1 in the next nucleosome.

At higher salt concentrations, the 10-nm fiber is further compacted into the 30-nm fiber.

By adding twists to make these nucleosomes and solenoid structures, the DNA is supercoiled

Loops of DNA are attached to a protein scaffold made up of several non-histone proteins.

This scaffold maintains the shape of a chromosome - even in the absence of histones. Concept 30 – Higher cells incorporate an ancient chromosome. different type of chromosome is in the mitochondria. mitochondrial (mt) chromosome contains genes involved in the process of oxidative phosphorylation — the production and storage of energy. Mitochondria are similar in size to bacteria, and the mt genome retains bacteria-like features. mt genome is a circular molecule. very few introns are found in mt genes. Plants another chromosome in the chloroplasts.

Concept 31 - Most DNA does not encode protein.

A large proportion of eukaryotic DNA is made of repeated sequences that do not encode proteins. Long non-coding sequences — or intergenic regions — separate relatively infrequent "islands" of genes. Numerous non-coding sequences — introns — are also found within genes, interrupting the protein-coding regions, or exons. It is estimated that only about one percent of human DNA encodes protein. About 25% make up genes and regulatory elements.

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Concept 32 - Some DNA can jump. 1950s, McClintock showed that certain DNA fragments, termed transposons, can be activated to transpose ("jump") from one position on a chromosome to another. McClintock thought that transposition provides a means to rapidly reorganize genes in response to environmental stress. The work was based entirely on observation of chromosomes and genetic crosses. This work led to the modern concept of chromosomes as dynamic, changing structures. Alu is an example of a so-called "jumping gene" - a transposable DNA sequence that "reproduces" by copying itself and inserting into new chromosome locations. Other animation and quizzes

• http://www.dnalc.org/resources/animations/alu.html

• http://highered.mcgraw-hill.com/sites/0072995246/student_view0/chapter23/mechanism_of_transposition.html

• http://highered.mcgraw-hill.com/sites/0072995246/student_view0/chapter23/simple_transposition.html

• http://highered.mcgraw-hill.com/sites/0072995246/student_view0/chapter23/transposons__shifting_segments_of_the_genome.html

Concept 33 - Genes can be turned on and off. Organisms can regulate gene expression. French researchers studied gene regulation using bacteria. When lactose is available, E. coli turn on a set of genes to metabolize the sugar. Lactose removes an inhibitor from the DNA. Removing the inhibitor turns on gene production. The gene that produces the inhibitor is a regulatory gene. Cells not only have genetic plans for structural proteins within their DNA, they also have a genetic regulatory program for expressing those plans.

First we will do a classroom test of the concepts.

Now we will look at the problem sets for these concepts.

You can work with a partner and enter your answers in socrative.com

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Summary concept map:

Before class: Look at the following concepts. Write down any words you don’t know. If you don’t understand the concept, please read the following notes carefully. Be prepared to ask questions in class. After we have discussed your questions, there will be a short test of these concepts in class. Concept 34 - Genes can be moved between species http://www.dnaftb.org/34 : http://www.dnaftb.org/34/animation.html Concept 35 - DNA responds to signals from outside the cell http://www.dnaftb.org/35 : http://www.dnaftb.org/35/animation.html Concept 36 - Different genes are active in different kinds of cells http://www.dnaftb.org/36 : http://www.dnaftb.org/36/animation.html Concept 37 - Master genes control basic body plans http://www.dnaftb.org/37 : http://www.dnaftb.org/37/animation.html Concept 38 - Development balances cell growth and death http://www.dnaftb.org/38 : http://www.dnaftb.org/38/animation.html

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Concept 34 - Genes can be moved between species. The genetic code is universal. The polymerases of one organism can accurately transcribe a gene from another organism. Eg, different species of bacteria obtain antibiotic resistance genes by exchanging small chromosomes called plasmids. 1970s - researchers used this type of gene exchange to move a "recombinant" DNA molecule between two different species. 1980s, other scientists adapted the technique and spliced a human gene into E. coli to make recombinant human insulin and growth hormone. Recombinant DNA technology — genetic engineering — makes it easier to study how genes work. If we can’t test gene function using animal models, genes can first be expressed in bacteria or cell cultures. Similarly, the phenotypes of gene mutations and how well drugs and other agents work can be tested using recombinant systems. Cohen and Boyer's recombinant DNA technique "created" the genetic engineering industry. In 1974, the technique was submitted for patenting, and in 1976, the first biotech company, Genentech Inc., was established based on recombinant DNA technology. Concept 35 - DNA responds to signals from outside the cell.

Cells have to communicate with each other so they can grow and develop. Also they need to react to signals that come from other parts of the body. Chemicals, eg. hormones released by various glands travel throughout the body to stimulate the growth of certain cell types. Cells which can be stimulated by a particular hormone have a specific receptor in the cell membrane. The binding of a hormone to its receptor starts a series of molecular transformations, called signal transduction, that relay the growth signal through the cell.

First, the receptor transduces the signal through the cell membrane to the internal membrane surface, where it activates protein "messengers." These messengers are the start of a cascade of chemical reactions, often involving the addition of phosphate groups. This is the signal that passes through the cytoplasm and into the nucleus. In the final step of signal transduction, DNA binding proteins attach to regulatory sequences and start or stop DNA replication or transcription.

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Concept 36 - Different genes are active in different kinds of cells. Different kinds of cells are specialized to perform different functions. But every cell of an organism has the same set of genetic instructions. Since biochemical function is caused largely by specific enzymes (proteins), different sets of genes must be turned on and off in the various cell types. This is how cells differentiate. This idea of cell-specific expression of genes is also shown by hybridization experiments that can identify the unique mRNAs in a cell type. DNA arrays and gene chips now allow us to quickly find all gene activity of an organism.

Some methods of DNA arrays and gene probes.

Hybridization is when single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA. Raising the temperature causes DNA molecules to separate into single strands. These strands are complementary to each other but may also be complementary to other sequences present in their surroundings. Lowering the surrounding temperature allows the single-stranded molecules to anneal or “hybridize” to each other.

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FISH (fluorescence in situ hybridization) is a cytogenetic technique that is used to detect and localize the presence or absence of specific DNA or RNA sequences on chromosomes or other parts of the cell. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH can also be used to detect and localize specific RNA targets (mRNA, lncRNA [Long non-coding RNAs] and miRNA) in cells, circulating tumor cells, and tissue samples.

Concept 37 - Master genes control basic body plans. The development of an organism — from a fertilized egg, through embryonic and juvenile stages, to adulthood — requires the coordinated expression of sets of genes at the proper times and in the proper places. Studies of several mutations in the fruitfly, Drosophila, helped understanding the molecular basis of development. Early embryonic genes express proteins that set up the orientation and define the body segments of the fly embryo. Then "homeotic" genes act on the segments to make different body parts. Homeotic genes from Drosophila and vertebrates share a 180-nucleotide region, the homeobox. Homeobox proteins have structures similar to the regions of regulatory proteins that bind to DNA promoters and enhancers.

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Homeotic proteins get coordinated expression when the protein binds to a specific promoter or enhancer sequence shared by a number of genes involved in the development of a body region or segment. Concept 38 - Development balances cell growth and death.

Growth is from the reproduction of new cells from pre-existing ones, by the process of cell division (mitosis). Once a tissue or organ reaches an appropriate size, mitosis slows and cells enter a resting phase. This cell cycle of growth and rest is controlled by "checkpoint" molecules first found in the 1980s and 1990s in yeast, and then in other eukaryotes. Normal development requires that some healthy cells be removed by a process called "apoptosis."

First ideas about apoptosis came from studies of the roundworm Caenorhabditis elegans. In C. elegans followed the development of each of the 959 cells in the adult can be traced from the fertilized egg. Analysis of cell "fates" showed that specific cells are programmed to die at specific times during embryonic development. Problems in the program can give too many cells — this is what happens in cancer.

First we will do a classroom test of the concepts.

Now we will look at the problem sets for these concepts.

You can work with a partner and enter your answers in socrative.com

Next we will look at the use of computers and other tools on a very large scale.

Before class: Look at the following concepts. Concept 39 - A genome is an entire set of genes www.dnaftb.org/39 : http://www.dnaftb.org/39/animation.html Concept 40 - Living things share common genes www.dnaftb.org/40 : http://www.dnaftb.org/40/animation.html Concept 41 - DNA is only the beginning for understanding the human genome www.dnaftb.org/41 : http://www.dnaftb.org/41/animation.html

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Concept 39 - A genome is an entire set of genes. The human genome is made up of a set of very long DNA molecules, one in each chromosome. There are an estimated 20,500 genes. The Human Genome Project determined the entire nucleotide sequence of each of these DNA molecules — and is trying to find the location and identity of all the genes. Sequencing the human genome has relied mainly on automated machines that sequence the DNA and computer programs that search and identify genes. Protein-coding sequences are a small fraction of the genome (approximately 1.5%), and the rest is associated with non-coding RNA molecules, regulatory DNA sequences, LINEs, SINEs, introns, and sequences for which as yet no function has been found. LINE - Long interspersed nuclear element SINE - Short interspersed nuclear element These are retrotransposons - genetic elements that can amplify themselves in a genome and are in the DNA of many eukaryotic organisms. Concept 40 - Living things share common genes. Nearly all living things have a genetic code in DNA and RNA. In evolution of new life, living things form new genes to support different body plans and types of nutrition. But complex organisms still have many genes that control core metabolic functions from their primitive past. Genes are kept during an organism's evolution; however, genes can also be exchanged or "stolen" from other organisms. Bacteria can exchange plasmids carrying antibiotic resistance genes through conjugation, and viruses can insert their genes into host cells. Some mammalian genes have also been adopted by viruses and later passed onto other mammalian hosts. However an organism gets and retains a gene, regions needed for the correct function of the protein are always conserved. Some mutations can accumulate in non-essential regions; these mutations give a history of the evolutionary life of a gene.

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Concept 41 - DNA is only the beginning for understanding the human genome. Although DNA transmits genetic information through time, it mostly has a passive role. Proteins encoded by DNA carry out the many cellular reactions that constitute "life." Now that we know about tens of thousands of genes, we are left with the question: "What do proteins made by these genes actually do?" Mutant organisms to give clues about protein function. Specific mutants can be created by inserting an altered or non-functioning copy of a gene back into a living organism, then looking for changes in behavior or development. Since mice breed quickly and share about 99% of their genes with humans, they have become the animal model of choice for large-scale functional studies. However, doing a single transgenic experiment is much, much more difficult than sequencing the gene itself. There is a lot of work needed if we want to understand the human genome.

First we will do a classroom test of the concepts.

Now we will look at the problem sets for these concepts.

You can work with a partner and enter your answers in socrative.com

Review questions will be provided.