BSC2010 Lectures 24-28
BSC2010 Lectures 24-28 BSC2010
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Date Created: 03/29/16
BSC 2010 Study Guide Lectures 24-28 Lecture 24: Regulation of Gene Expression in Bacteria - Understand that regulation of gene expression means regulation of the production of protein product of the gene. o Basically, all genetic codes are the same, but depending upon how the gene is expressed is how it is portrayed in an organism. For example, we all have the same genetic code but we all look different, act different, and behave differently because of the way our genes are expressed and how the production of the protein products from the gene are regulated. o Another definition: the appearance in a phenotype of a characteristic or effect attributed to a particular gene. - Understand that bacteria regulate gene expression by controlling transcription. o A bacteria cell regulates gene expression by controlling the rate at which DNA is converted to RNA. o A transcription factor is used which is a substance, such as a protein, that contributes to the cause that contributes to a specific biochemical reaction or bodily process. o Transcription regulation has vital components in order to make it work including the promoters and operators. A promoter is an element of DNA that may bind to RNA polymerase and other proteins for the successful initiation of transcription directly upstream of the gene. Operators recognize repressor proteins that bind to a stretch of DNA and inhibit the transcription of the gene. o Overall, transcription regulation basically just means that a bacteria cell is able to stop and start transcription as needed to perform basic cell activity or code for certain proteins by using the described components above. - Be able to explain what an operon is and how operons differ from typical genes. o An operon is a unit made up of linked genes that is thought to regulate other genes responsible for protein synthesis. o Another definition: An operon contains an operator, a common promoter, and one or more structural genes, which is controlled as a unit to produce messenger RNA (mRNA), in the process of transcription by an RNA polymerase. o For instance in the Lac Operon, when the Lac Operon is activated, all of the genes necessary to create components necessary to breakdown lactose are in this operon. Basically, it is a set of necessary genes to accomplish the task of that operon. o A repressible operon is normally on and is repressed in order to constrict the amount of product. An inducible operon is usually off and will have to be activated in order to be transcribed. o Another example using the Lac operon: The purpose of the operon is to keep these genes turned off If there is no lactose around, and to turn them on when lactose is plentiful. The lac operon contains three genes which code for enzymes necessary for the metabolism of the sugar lactose. o - Regulation of trp operon by tryptophan, trp repressor and trp operator. o The trp operon is repressed when tryptophan levels are high by binding the repressor protein to the operator sequence via a corepressor which blocks RNA polymerase from transcribing the trp-related genes. o The trp operon is activated when tryptophan levels are low by dissociation of the repressor protein to the operator sequence which allows RNA polymerase to transcribe the trp genes in the operon. - Lac Operon and its regulation by lactose and glucose – lac repressor and CAP protein. o When glucose is present, cyclic AMP is low, CAP does not bind to the activator binding site, and transcription is turned off. o The lac repressor binds to the operator and prevents transcription. o When both glucose and lactose are present, transcription is turned off. o In the absence of glucose, when lactose is present it combines with the repressor, allowing RNA polymerase to carry on transcription. o When glucose levels decline in E. coli, catabolite activator protein (CAP) is bound by cAMP to promote transcription of the lac operon. - trp operon is said to be repressible and the lac operon is said to be inducible. o The lac operon is said to be inducible because when allolactose is present, it induces the inactivation of the lac repressor. o The trp operon is said to be repressible because the presence of tryptophan causes a conformational change which allows the repressor to bind to the operator, thus blocking transcription. - Transcription factors are proteins that regulate the ability of RNA polymerase to bind the promoter and initiate transcription, activators and repressors. o Transcription factors are an important component in gene expression and regulation. Lecture 25: Regulation of Gene Expression in Eukaryotes - Transcription regulation via transcription factors is important but eukaryotes also regulated the packing (coiling) of chromatin, and the processing of mRNA. o Difference in eukaryotic and bacteria gene expression: bacteria regulates gene expression only through transcription regulation whereas eukaryotes regulate gene expression via transcription regulation (by use of transcription factors) as well as packaging/ coiling of chromatin, and the processing of mRNA. - Understand that each cell type in multicellular eukaryotes uses a different subset of its genes, each cell type makes a characteristic set of proteins. o The genetic code is all the same, but each subset of genes (coded for by the genetic code) makes a different type of protein in order to make the polypeptide chain which will be used in different processes. This goes back to the definition and importance of gene expression. - Chromatin – DNA plus histone proteins. Tightly packed chromatin – no transcription. Acetylation of histone proteins – loosely packed chromatin, active transcription. o Histones are small groups of basic proteins used to make chromatin. The chromatin coils and loops up in order to form into a more organized package for use. o If the chromatin is too tightly packed, transcription cannot occur. This is where acetylation comes in handy. Acetylation of histone proteins loosens the packed chromatin, therefore activating transcription. - Alternate Splicing o Alternative splicing is a regulated process during gene expression that results in a single gene coding for multiple proteins. In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene. o o Textbook definition: A type of eukaryotic gene regulation at the RNA- processing level in which different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns. - Hormones can regulate transcription: o Hormones can regulate transcription in eukaryotic organisms. Lecture 26: Viruses - Viruses are not considered living organisms. - Basis structure of viruses and viral genomes. o The protein layer that surrounds and protects the nucleic acids is called the capsid. When a single virus is in its complete form and has reached full infectivity outside of the cell, it is known as a virion. A virus structure can be one of the following: icosahedral, enveloped, complex or helical. o o Viral genomes: It is encoded either in DNA or, for many types of virus, in RNA. A virus has either DNA or RNA genes and is called a DNA virus or a RNA virus. The genome includes both the genes and the non- coding sequences of the DNA/RNA. - Know that a virus’ genome must, at a minimum, code for the virus’ coat proteins and they may also have to code for specialized DNA or RNA polymerase enzymes needed for viral replication (particularly RNA viruses). - Generalized life cycle of a DNA virus, an RNA virus that replicates by transcription, and a retrovirus. o Viral replication is the formation of biological viruses during the infection process in the target host cells. Viruses must first get into the cell before viral replication can occur. From the perspective of the virus, the purpose of viral replication is to allow production and survival of its kind. By generating abundant copies of its genome and packaging these copies into viruses, the virus is able to continue infecting new hosts. Replication between viruses is greatly varied and depends on the type of genes involved in them. Most DNA viruses assemble in the nucleus while most RNA viruses develop solely in cytoplasm. o Retrovirus – virus made through reverse transcription. Any of a group of RNA viruses that insert a DNA copy of their genome into the host cell in order to replicate, e.g., HIV. A retrovirus contains RNA instead of DNA. o HIV is an RNA virus that replicates by transcription. o A typical virus reproductive cycle: Entry and uncoating Replication of viral DNA or RNA Transcription and synthesis of coat protein(s) Assembly of viral particles and escape from the host cell - How does replication of a membrane encapsulated virus differ from a virus that lacks a membrane envelope? o Makes it easier for the virus to attach to the host cell because it does not have to go through the uncoating process before attaching. The capsid encloses the genetic material of the virus. o If a virus has an outer membrane, it has a capsid (the outer membrane is the capsid). - Reverse Transcriptase o A DNA polymerase that uses an RNA template. Lecture 27 – Recombinant DNA and Gene Cloning - Restriction Enzymes: o Different restriction enzymes cut different DNA sequences. They are essential tools for recombinant DNA technology. The enzyme "scans" a DNA molecule, looking for a particular sequence, usually of four to six nucleotides. o The site at which the restriction enzyme cuts is known as the “restriction site.” o The DNA of a bacterial cell is protected from the cell’s own restriction enzymes by the addition of methyl groups (--CH3) to adenines or cytosines within the sequences recognized by the enzymes. o The restriction sites form a ‘sticky end’ which is where the complementary base pairing will bind from the new DNA fragment. - Plasmids: o A plasmid is a small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. Plasmids naturally exist in bacterial cells, and they also occur in some eukaryotes. Often, the genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance. o In gene cloning, DNA fragments are randomly inserted into plasmids. Recombinant plasmids are randomly put into bacteria. o - Recombinant DNA: o DNA that has been formed artificially by combining constituents from different organisms. o i.e., inserting calf liver DNA into bacterial DNA to form plasmid - Outline of Cloning: o Cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. o Scientists use cloning vectors to carry recombinant DNA. o Cloning is used to express eukaryotic genes of interest. - Gene libraries, genomic vs cDNA libraries, use of reverse transcriptase in making cDNA: o A “gene library” is a collection of cloned DNA fragments each in a bacterial colony. o A genomic library consists of DNA cloned pieces of genomic DNA (chromosomes). o A cDNA library consists of cloned mRNAs. o Reverse transcriptase is an enzyme that allows for the “backwards” flow of information from RNA to DNA.. so cDNA is complementary DNA and it is formed from mRNA by use of reverse transcriptase. - The steps in PCR: o PCR (polymerase chain reaction) makes many copies of a DNA sequence by repeated rounds of DNA replication. o First, the DNA is denature (unwound), then the short single stranded DNAs (PRIMERS) are allowed to bind – this is the annealing step. DNA polymerase extends from the primers. Each PCR cycle doubles the number of target molecules, so cycle 2 yields 4 molecules, cycle 3 yields 8 and so on. (2^ # of cycles) o FULL STEPS IN PCR: 1) Denaturation: heat briefly to separate DNA strands 2) Annealing: cool to allow primers to form hydrogen bonds with ends of target sequence 3) Extension: DNA polymerase adds nucleotides to the 3’ end of each primer. o - Electrophoresis of Nucleic Acids: o Gel electrophoresis separates DNA molecules based on their size. - Dideoxy nucleotide sequencing of DNA: o Allows for the analysis of the gene at a nucleotide level. o Lecture 28 - Biotechnology Applications: - Cloning of genes for human proteins into bacteria, for production of important pharmaceuticals: o Scientists can manipulate cloning of genes in order to produce a gene that will be more or less responsive to certain pharmaceutical drugs. - Know that transgenic organisms – cloned genes inserted into chromosomes of the target organism. The insertion of the plasmid occurs at random sites in the chromosome, can cause mutations if it inserts in an important gene.
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