* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download CHAPTER 16 – THE MOLECULAR BASIS OF INHERITANCE
Transposable element wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Epigenetics wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
Minimal genome wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Gene expression profiling wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Human genome wikipedia , lookup
Genome (book) wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
Genomic library wikipedia , lookup
Genome evolution wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Genetic engineering wikipedia , lookup
Oncogenomics wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
DNA vaccination wikipedia , lookup
DNA supercoil wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Molecular cloning wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Epigenomics wikipedia , lookup
Genome editing wikipedia , lookup
Designer baby wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Primary transcript wikipedia , lookup
Point mutation wikipedia , lookup
Non-coding DNA wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Microevolution wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Helitron (biology) wikipedia , lookup
UNIT 5 – MOLECULAR GENETICS (CHAPTERS 16 – 20) CHAPTER 19 – EUKARYOTIC GENOME YOU MUST KNOW: The impact of DNA methylation and histone acetylation on gene expression The role of oncogenes, proto-oncogenes, and tumor suppressor genes in cancer. I. CHROMATIN STRUCTURE Eukaryotes have substantially more DNA than prokaryotes. This DNA must be organized and managed for cell specialization. Gene expression in both groups is regulated mostly during transcription. Eukaryotic DNA is packed with proteins. The complex of these two molecules is called chromatin. During interphase the chromatin is loose, extended. At the beginning of cell division, this extended chromatin undergoes a coiling and folding process that significantly shortens the chromatin into chromosomes. Each chromosome contains a single, linear DNA double helix. (The total length of DNA in each human cell is about 99 cm = 3 feet). First level of DNA packaging: histones, small, bead-shaped proteins with positively charged amino acids can bind to the negatively charged DNA molecule and wrap the DNA double helix up like a spool. Each histone with the wrapped DNA is a nucleosome. The nucleosome basically remains intact throughout transcription and translation. Second level of DNA packaging: The neighboring nucleosomes and the linker DNA between them will interact with each other. This pulls the nucleosomes closer together. This is the form that is seen during interphase. Next level of packaging: A long protein scaffold made up of nonhistone proteins folds the second level protein fiber up by forming loops. These loops may help to organize the chromatin that is being actively transcribed. The organization of the chromatin in this phase is visible under light microscope. The darker colored and compact heterochromatin is not transcribed as opposed to the actively transcribed euchromatin that is lighter in coloration. During metaphase the chromatin folds further, resulting in the maximally compacted chromosome with two chromatids. Animation: http://www.dnai.org/text/mediashowcase/index2.html?id=564 I. GENE REGULATION DURING TRANSCRIPTION All organisms have mechanisms for turning genes on or off. This process either activates the genes for transcription or inactivates them into a dormant stage. Multicellular organisms also undergo long-term gene regulation that result in cell differentiation (Think back to the genetic update conference) Animation (shows a brief review of what we learned so far in this unit and how it relates to gene expression): http://www.genomicseducation.ca/animations/gene_expression.asp A. Differential Gene Expression Although all cells in a multicellular organism have the same genome, each type of specialized cell expresses a different set of genes to carry out its specific function – differential gene expression. Because only a small percentage of the genome is coding gene (gene that codes the information for a polypeptide) transcription proteins must locate the right genes at the right time to transcribe them. Gene expression can be regulated at various points in eukaryotic cells. On these points (shown below) gene expression can be turned on, off, accelerated or slowed down. Great overview of gene expression: http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter18/animations.html# B. Regulation on the DNA level (Chromatin Structure Regulation) The organization of the chromatin in the chromosome is important in gene expression. Heterochromatin that is tightly packed, is usually not expressed. Chemical modifications to the histones or to the DNA can influence chromatin structure and gene expression: a. Histone acetylation and deacetylation – adding acetyl groups to histones does not allow them to bind to each other any more. As a result, chromatin has a looser structure and transcription factors have easier access to the genes of the acetylated region. Acetylation promotes transcription. b. DNA methylation – methyl groups can be attached to the DNA molecule at certain bases (usually cytosine). Highly methylated DNA is usually inactive. Methylation also seems to be responsible for genomic imprinting – permanent inactivation of the mothers or fathers genes for certain traits. C. Regulation of Transcription Initiation The eukaryotic gene and additional control DNA elements are responsible for adding the proper transcription factors to the promoter region of the DNA molecule before the RNA polymerase II can bind to it. Figure 19.5 A. Post-Transcriptional Regulation: Once the gene has been transcribed, mRNA processing, mRNA degradation and translation also can change the outcome of the protein synthesis: a. RNA processing – This type of regulation is only available in eukaryotes. One example of alternating the primary transcript is alternative splicing – different RNA molecules are produced from the same primary transcript, depending on which segments are treated as exons and introns. Alternative splicing is controlled by regulatory proteins. b. mRNA degradation – mRNA molecules are short lived and degrade very quickly in prokaryotes. However, in eukaryotes mRNA molecules can survive for weeks and translated repeatedly in the cytoplasm of some cells. Eukaryotic mRNA molecules can be inactivated by using other RNA molecules (miRNA, siRNA) and protein complexes. These complexes can inactivate or degrade the mRNA molecule. c. Initiation of Translation –Regulatory proteins can bind to the untranslated region of the mRNA and can block translation. d. Protein processing or degradation – phosphate groups can be added to inactive forms of already made proteins to activate them or phosphate groups can be removed to inactivate proteins. Proteasome molecules can degrade already existing proteins. II. THE GENETICS OF CANCER A. Types of Genes Associated with Cancer Cancer can be caused by mutations that occur in genes that are responsible for cell growth and division (genes that code for growth factors, growth factor receptors and intracellular molecules of cell signaling) These mutations can be random or caused by mutagens in the environment or certain viruses (HPV cause cervical cancer, EpsteinBarr virus causes infectious mononucleosis). All tumor viruses transform cells into cancerous cells by integrating viral DNA into the host DNA. Proto-oncogenes are normal genes that are responsible for cell growth and differentiation in animals and humans. These genes can become mutated and become oncogenes (cancer causing genes). There are three main groups of genetic changes that can convert proto-oncogenes into oncogenes: a. A mutation in the gene can change the protein structure that the gene codes for. The produced protein becomes overactive or loses its sensitivity to regulators and does not degrade fast enough. b. The gene can have more than normal number of copies by gene amplification. This result in an increased concentration of proteins that are produced by the proto-oncogene. c. Translocation or transposition of the gene can put it in new locations under new genetic control. This can result in an overproduction of the coded protein. d. A point mutation can also occur in the control element of the gene, “upstream” from the gene and can result in excess amount of protein. Figure 19.11 Tumor-suppressor genes – Genes in cells that normally inhibit cell division. The proteins that these genes code, help to prevent uncontrolled cell growth. Any mutation that results in the decrease of the activity of the tumor-suppressor genes can cause cancer. According to today’s theory of cancer development, more than one somatic mutation is needed to produce all the changes that result in cancer. These mutations usually accumulate over time, so the rates of cancer increases as a person ages. Steps of developing colorectal cancer: o First, the loss of a tumor-suppressor gene can result in small benign tumors (polyps) o Second, a new mutation results in the overproduction of a protein that stimulates cell division. The result is a larger benign growth (adenoma) of the same cells. o Third, a serious mutation in a tumor-suppressor gene can result in a malignant tumor (carcinoma) in the same cells. Figure 19.13 Certain cancers can run in families because a person can inherit an already mutated gene that starts the person on the pathway of many other mutations that can lead to cancer sooner. Examples of cancer types that be the result of inherited mutations are some colorectal cancers and some breast cancers. Movie segment: http://www.pubinfo.vcu.edu/secretsofthesequence/playlist_frame.asp III. NONCODING DNA SEQUENCES, TRANSPOSONS The bulk of most eukaryotic chromosomes are made up of noncoding DNA sequences, often described as “junk DNA”. However, there is evidence today that these sequences play important roles in the cell. Trends in genomic complexity shows that as we move up on the phylogenetic tree, more complex organisms usually have more DNA but fewer coding genes in a given length of DNA. In prokaryotes, most genes code for proteins, tRNA and rRNA and there are a few noncoding, regulatory sequences, such as promoters. Coding occurs continuously without interruptions. In eukaryotes, most of the DNA does not code proteins or RNA and it includes more complex regulatory sequences. Some of these noncoding sequences are present as introns within genes, a large portion of DNA is also made up of gene fragments, mutated genes that are not functional. Most of the intergenic DNA is repetitive DNA, sequences that are present in multiple copies in the genome. About 44% of these sequences are transposable elements and related sequences. Eukaryotic transposable elements are two kinds: o Transposons – sequences that move around the genome by means of a DNA intermediate. These sequences can move by “cut-and-paste” mechanisms that remove them from one site of the genome and inserts them at another site. Transposons can cause damage to functioning genes but also can give rise to new variations of proteins by changing the arrangements of genes. o Retrotransposons – these are mobile genetic elements that always leave a copy at the original site during transposition because it is copied into an RNA molecule. The RNA molecule than copied into a DNA by reverse transcriptase and than inserted back into the genome as DNA at another location. These can also result in new genetic arrangements that are usually harmful but can have evolutionary significance. Figure 19.16 CHAPTER 20 – DNA TECHNOLOGY AND GENOMICS YOU MUST KNOW: The terminology of biotechnology The steps in gene cloning with special attention to the biotechnology tools that make cloning possible. The key ideas that make PCR possible How gel electrophoresis can be used to separate DNA fragments or protein molecules. I. Understanding and Manipulating Genomes: Biotechnology is a booming field of science with constantly improving technology and new discoveries weekly. Some key terms to know: o Recombinant DNA – DNA in which nucleotide sequences from two different sources, often different species, are combined in vitro into the same DNA molecule. o Genetic engineering – the direct manipulation of genes for practical purposes o Biotechnology – the manipulation of organisms or their components to make useful products (from wine and cheese making to analyzing personal genomes and fixing mutations) Radiolab: http://www.wnyc.org/flashplayer/player.html#/play/%2Fstream%2Fxspf %2F92351 – 35:00 min II. DNA Cloning: A. The Method of DNA Cloning: Gene cloning – methods for preparing well-defined, gene-sized pieces of DNA in multiple identical copies. Most commonly bacteria and their plasmids are used: o Plasmid is isolated o Foreign DNA is inserted into the plasmid – recombinant DNA o Plasmid is returned into the bacterium o Bacterium reproduces to form clones of identical cells Cloned bacteria can make many copies of a certain gene and can produce certain proteins. B. Using Restriction Enzymes: Restriction enzymes – enzymes that cut DNA molecules at a limited number of specific locations. In nature, these enzymes protect the bacterial cell against intruding DNA from other organisms, by cutting this DNA segments up. Restriction sites – short segments of DNA that are recognized by the restriction enzyme. The bacterium’s own DNA is protected by methylation from restriction enzymes. http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations.html# C. Cloning a Eukaryotic Gene in a Bacterial Plasmid The original plasmid is called a cloning vector – this plasmid has the ability to carry foreign DNA into a cell and replicate it there. Bacterial plasmids are widely used cloning vectors, because they are easy to isolate, manipulate and can be reintroduced back into the bacterium after isolation. Because bacterial cells reproduce quickly, the inserted gene or its proteins can be obtained in large quantities. Use: http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations.htm l# After genes are inserted into bacteria by using DNA cloning, the success of the experiment can be analyzed by two methods: a. Looking for the inserted gene in the bacterial colonies b. Looking for the synthesized proteins in the new bacterial colonies Nucleic acid hybridization – is the process that detects certain sequences of the DNA molecule by using nucleic acid probes that are radioactively labeled. These probes can bind to denatured DNA molecules (two strands of DNA separated) and radioactively label the colonies that contain the inserted gene. Animations: DNA cloning: http://www.sumanasinc.com/webcontent/animations/content/plasmidcl oning.html http://highered.mcgrawhill.com/sites/0072556781/student_view0/chapter14/animation_quiz_1.htm l Restriction enzymes: http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations.html# III. THE POLYMERASE CHAIN REACTION (PCR) When the source of DNA molecule is impure or there is only a small amount of DNA present, PCR is the most effective way to amplify one or many segments of DNA. This method can make millions of copies of a segment of DNA in a few hours. PCR is a three-step cycle that brings about a chain reaction that will produce an exponentially growing number of copies of identical DNA molecules. Steps: a. first the target sequence is denatured (separated to individual polynucleotide chains) by heat b. second, cooling allows short segments of DNA primers to attach by hydrogen bonding at the 5’ → 3’ direction c. Heat-stable DNA polymerase is used to assemble the nucleotides of the new strands Animations: PCR -- http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations.html# http://www.dnalc.org/ddnalc/resources/pcr.html PCR song: http://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/ IV. GEL ELECTROPHORESIS This process uses a gel to separate various segments of DNA molecule or protein based on their size and electrical charge. A mixture of DNA segments of different sizes can be injected into the wells of the gel than put in an electric current. The electric current is running from the – to the + electrode and drags the molecules with it. The smallest pieces of molecules run the furthest. A fluorescent dye can be used to dye the DNA segments and make them visible. Gel electrophoresis can be used to locate mutations on various DNA molecules, separate certain segments of DNA from the others for further examination, purify DNA, the band pattern can help in identifying a person etc. Interactive lab: http://learn.genetics.utah.edu/content/labs/gel/ Animation: http://www.dnalc.org/ddnalc/resources/electrophoresis.html V. SOUTHERN BLOTTING This technique combines gel electrophoresis and DNA hybridization to allow researchers to find a specific human gene. It can be used to identify individual nucleotide differences (mutations) in the DNA molecule. It can also compare particular DNA fragments from different sources that were digested by restriction enzymes. http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations.html# Biotechnology movie clips: http://www.pubinfo.vcu.edu/secretsofthesequence/playlist_frame.asp THIS IS THE END OF THE MOLECULAR BIOLOGY UNIT