CH 8: Microbial Genetics

How would you create a genetically modified plasmid?

1) Select gene 2) Cuts produce DNA fragment with 2 sticky ends and 2 fragments join by base pairing. 3) joined fragments make linear or circular molecules 4) DNA ligase used to join backbones of fragments producing molecule of recombinant DNA. 5) Reproduce through Binary Fission

Mechanisms of Gene Control (of transcription) in Eukaryotes.

1) Transcriptional Control 2) Post-Transcriptional Control 3) Translational Control 4) Post-translational Control

How does transduction occur?

1) Virus infects bacterial cell and replicates its genome within, leading to cell lysis 2) Virus takes up own DNA again, with some viruses taking in lysed bacterial DNA segments 3) Bacterial DNA is inserted into new cell by virus and integrates into nucleus

What are the steps of translation?

1. Initiation: mRNA binds to the smaller subunits of the ribosome, followed by the burning of the larger subunit. The first tRNA binds to the start codon. 2. Elongation: a tRNA carrying and amino acid attaches to the mRNA at the P-site Of the ribosome. The anti-codon of the tRNA matches the codon of the mRNA. A second tRNA attaches to the mRNA in the A-Site. The mRNA shifts, moving the tRNA in the P-site out (detaches from the mRNA) and the tRNA in the A-site into the P-site. The amino acids carried by the tRNAs are linked together by a peptide bond and the first tRNA leaves without an amino acid (it will find another). This leaves room for a third tRNA to enter the A-site. The process continues until a stop codon on the mRNA is read. 3. Termination: When a stop codon is reached, the ribosome breaks apart into small and large subunits again - the mRNA and the protein are released and float away

How do you make a DNA fingerprint?

1. PCR 2. Restriction enzymes 3. load wells 4. gel electrophoresis 5. stain 6. UV light 7. DNA fingerprint 8. confirm presence at the scene

What are the methods of gene transfer or bacterial recombination in prokaryotes?

1. Transformation **(5) Transformation by Plasmid 2. Conjugation 3. Transformation by Phage (virus) 4. Transposons "jumping genes"

What are 4 different ways that bacteria can transfer genes horizontally?

1. Transformation 2. Transformation by Plasmid 3. Plasmid 4. Conjugation

What is a competent cell?

A cell that has been modified to take up DNA from the environment via transformation

What is a polyribosome?

A cluster of ribosomes translating the same mRNA, but positioned at different sites along the mRNA

What is an operon and what are its parts?

A set of operator and promoter sites and the structural genes they control define and OPERON. Thus, the combination of three lac structural genes and the adjoining control regions is called the lac operon.

Alleles

Alternate forms of a gene at the same locus

repressible operon

An operon under negative control. It is usually "on" but can be turned "off".

inducible operon

An operon under positive control. It is usually "off" but can be turned "on".

Horizontal Gene Transfer

Bacteria can pass their genes not only to their offspring, but also laterally, to other microbes of the same generation. It occurs in several ways. In all of the mechanisms, the transfer involves a DONOR CELL that gives a portion of its total DNA to a RECIPIENT CELL. Once transferred, part of the donor's DNA is usually incorporated into the recipient's DNA; the remainder is degraded by cellular enzymes. The recipient cell that incorporates donor DNA into its own DNA is called a recombinant.

What happens when mRNA is processed?

Before mRNA can be used by ribosomes as a template for building proteins, it must first be processed. Key steps are the addition of a methylated cap and a poly-adenylated tail. Involved in the processing are RNA polymerase, cleavage factors, and Poly A polyermase. Processing of mRNA, begins with transcription. Soon after RNA polymerase being transcription, a methylated cap is added to the 5' end. Transcription then continues to completion. Following completon, RNA polymerase release the capped strand of pre-mRNA. Specific nucleotide sequences in the mRNA are bound by cleavage factors. The 3' end of the mRNA is next moved into the correct configuration for cleavage. Stablizing factors are then added to the complex. Poly A polymerase now binds to the mRNA and cleaves the 3' end. The complex beings to dissociate, and the cleaved 3' end quickly degrades. Poly A polymerase now synthesizes the polyadenylated tail, by adding adenine residues to teh cleavage site. Additional proteins then bind to the tail, incresing the rate at which is grows. When the tails reaches its full length, the poly A polymerase is singaled to stop adding residues, and the polyadenylation process is now complete. The processed mRNA is now ready to undergo splicing in preparation for translation.

Replication in Bacteria

Begins at the origin of replication (A specific site). Move bi-directionally around the chromosome with two replication forks moving in opposite directions away from the origin of replication. Results in two identical circular daughter chromosomes.

Mutations

Change in the BASE sequence of DNA. When the gene for an enzymes is mutated it is either inactive, less active, or, rarely, more efficient. This may be disadvantageous, lethal, or advantageous for the organism.

What is a codon and an anti-codon?

Codon: the language of mRNA is in the form aid codons, groups of 3 nucleotides. The sequence of codons on an mRNA molecules determines the sequence of amino acids that will be in the protein being synthesized. Each codon "codes" for a particular amino acid. This the genetic code. Anticodon: Each tRNA molecule has an anti-codon, a sequence of three bases that is complementary to a codon. In this way, a tRNA molecule can base-pair with its associated codon. "Recognize" a specific codon on the mRNA strand.

Which type of bacterial recombination involves a pili?

Conjugation

Types of Plasmids

Conjugative Plasmid: carries genes for SEX PILI and TRANSFER of the plasmid. Dissimilation Plasmids: encode enzymes for CATABOLISM of unusual compounds R factors: encode ANTIBIOTIC RESISTANCE code for BACTERIOTOXINS: proteins toxic to other organisms.

Transcription

Copying of a DNA sequence into and RNA sequence

Replication

Copying process by which a cell duplicates its DNA

Transcription: mRNA processing in Eukaryotes

DNA is made of introns and exons. Introns: segments between genes which do NOT code for proteins. Exons: segments of gene which code for proteins. During transcription, both introns and exons are transcribed into mRNA. Before leaving the nucleus, the INTRONS are spliced out leaving only the EXONS. Exact role of INTRONS is unknown. THIS DOES NOT HAPPEN IN PROKARYOTES! they do not have introns/exons In Eukaryotes, after TRANSCRIPTION, the processed mRNA exits the nucleus and enters the cytoplasm.

Why is proofreading so important in bacteria?

DNA polymerases are the enzymes that build DNA in cells. During DNA replication (copying), most DNA polymerases can "check their work" with each base that they add. This process is called proofreading. If the polymerase detects that a wrong (incorrectly paired) nucleotide has been added, it will remove and replace the nucleotide right away, before continuing with DNA synthesis

Central Dogma

DNA undergoes Replication where DNA is split and 2 old strands used as templates for the 2 new strands , semi conservative. DNA then undergoes TRANSCRIPTION by copying of a DNA sequence into an RNA (mRNA) sequence. The now formed mRNA undergoes TRANSLATION where mRNA transcripts are decoded into PROTEINS

Which bases are found in DNA, which in RNA?

DNA: Adenine---Thymine and Guanine---Cytosine RNA: Adenine---Uracil and Guanine---Cytosine

What is the general structure of a nucleotide (RNA and DNA)?

DNA: made of chains of nucleotides. Nucleotide has 3 parts: 1. BASE - nitrogen containing (Adenine, Thymine, Guanine, Cytosine). 2. 5-carbon SUGAR = deoxyribose 3. PHOSPHATE group To form a chain, nuclelotides are linked together with the sugar of one nucleotide attached to the phosphate group of the next with the bases hanging of the side. Forming a sugar phosphate backbone. The structure is that of an entwined DOUBLE HELIX with the nucleotides of opposite sides of the chain linked together with H-bonds in a ladder-like manner. It is the sequence of these bases that makes every organism unique. in 1951, WATSON and CRICK were credited with determining the structure of DNA.

Transfer of genetic material in Prokaryotes (bacteria):

DONOR CELL to a RECIPIENT CELL (making is RECOMBINANT CELL)

What is the difference between F+ cells and HFR cells?

Donors carrying F factors (F+ cells) transfer the plasmid to recipients (F- cells), which become F+ cells as a result. In some cells carrying F factors, the factor integrates into a chromosome, concerting the F+ cell to an Hfr cell. (high frequency of recombination). When conjugation occurs between an Hfr cell and an F- cell, the Hfr cell's chromosome (with its integrated F factor) replicates, and a parental strand of the chromosome is tranferred to the recipeint cell. ...By conjugation with an Hfr cell, an F- cell may acquire new versions of chromosomal genes (just as in transformation). However, it remains an F- cell because it did not receive a complete F factor during conjugation.

What is semiconservative replication?

Each of the parent nucleotide strands remains intact (conserved), but they are now bound to new nucleotide strands instead of each other.

Mistakes of DNA replication in bacteria

Excellent Quality control Rare but one of every 10^10 based incorporated may have a mistake.

What is a chromosome? What is a gene?

GENE: segments of DNA (RNA in some viruses) which code for functional products (PROTEIN). CHROMOSOME: structures containing DNA that physically carry hereditary information; the chromosome contains the genes; it consists of DNA and PROTEIN (histones in eukaryotes).

What is the correct DNA sequence to match CCGATTGCG?

GGCTAACGC

What is the correct sequence of mRNA to go with CCGATTGCG?

GGCUAACGC

What is the difference between genotype and phenotype?

Genotype: genetic makeup of an organism; refers to the GENES of an individual; the information that codes for all the particular characteristics of the organism. It represents POTNETIAL properties, but not the properties themselves. Phenotype: refers to the ACTUAL, EXPRESSED properties, such as the organism's ability to perform a particular chemical reaction. It is the manifestation of the genotype.

What is the function of helicase (unwinding enzyme), DNA polymerase, RNA polymerase, and ligase?

Helicase: unwinds double stranded DNA. DNA polymerase: synthesizes DNA; proofreads and repairs DNA. RNA polymerase: copies RNA from a DNA template DNA ligase: makes covalent bonds to join DNA strands; joins Okazaki fragments in new segments in excision repair

How does a bacteria overcome the problem of DNA replication being too slow for rapid growth requirements? (i.e., what are multiple replication forks?)

Horizontal Gene Transfer: During replication, bacterial cells undergo recombination into a recombinant cell between cells of the same generation.

DNA comes apart for copying along:

Hydrogen Bonds

Control of Gene Expression

In Eukaryotes: all the cells in an individual have EXACTLY THE SAME genes; So how can we have so many different types of cells? **different cells have different genes being actively EXPRESSED (i.e., muscle cells, nerves, adipose tissue). In Prokaryotes: Simpler, there is only one cell. No barrier between transcription and translation because they do not have a NUCLEUS.

Translation

In bacteria, there is no nucleus so TRANSLATION of the mRNA occurs while TRANSCRIPTION is occurring! Translation of mRNA begins at the START CODON (AUG). Translation ends at a STOP CODON: UAA, UAG, UGA

How is bacterial DNA different from human DNA?

It is a SINGLE, CIRCULAR DNA molecules associated with bacterial proteins. It's ONE chromosome is looped, coiled, and folded on itself and attached to the PLASMA MEMBRANE in at least one point.

During DNA replication which strand is synthesized discontinuously?

Lagging Strand

Constitutive vs. Regulated Genes

Many genes (60-80%) are CONSTITUTED - products CONSTANTLY being produced (genes always "ON"). **Code for enzymes needed in large quantities (i.e. glycolysis enzymes). The other 20-40% of genes are REGULATED. **Products produced only when needed ("OFF")!

Conjugation

Mediated by one kind of PLASMID (a circular piece of DNA that replicates independently from the cell's chromosome. However, plasmids differ from bacterial chromosomes in that the genes they carry are usually not essential for the growth of the cell under normal conditions. The plasmids responsible for conjugation are transmissible between cells during conjugation. Conjugation differs from transformation in 2 ways: 1) Conjugation requires DIRECT CELL-TO-CELL CONTACT. 2) the conjugating cells must generally be of OPPOSITE MATING TYPE; donor cells (F+) must carry the plasmid, and recipient cells (F-) usually do NOT. In G- bacteria: the plasmid caries genes that code for the synthesis of SEX PILI, projections from the donor's cell surface that contact the recipient and help bring the 2 cells into direct contact. G+ bacterial cells: produce sticky surface molecules that cause cells to come into direct contact with each other. In the process of conjugation, the plasmid is replicated during the transfer of a single-stranded copy of the plasmid DNA to the recipient, where the complementary strand is synthesized.

What are complementary base pairs and why are they important to DNA replication?

Nitrogenous bases can only bind with each other A to T and C to G. Complementary base pairing is very important in the conservation of the base sequence of DNA. This is because adenine always pairs up with thymine and guanine always pairs up with cytosine. As DNA replication is semi-conservative (one old strand an d one new strand make up the new DNA molecules), this complementary base pairing allows the two DNA molecules to be identical to each other as they have the same base sequence. The new strands formed are complementary to their template strands but also identical to the other template. Therefore, complementary base pairing has a big role in the conservation of the base sequence of DNA.

During translation, the first amino acid is brought into the:

P-site

What is a plasmid? What types are there?

Plasmids are extrachromosomal bits of DNA. Types: conjugative, dissimilation, R factors, and those that code for bacteriotoxins.

Translation

Process by which mRNA is decoded and a protein is produced

What is the difference between a repressible and inducible model?

REPRESSION: mechanism of INHIBITING gene expression. Responds to an overabundance or build up of the end-products from metabolic pathways. ***involves REPRESSORS (regulatory proetsin) which BLOCK RNA polymerase from transcription of the targeted genes. INDUCTION mechanism of "turning-on" the transcription of a gene. Involves INDUCERS (regulatory proteins). Induces the transcription of genes needed in special circumstances (i.e., the enzymes need for lactose or starch metabolism).

General facts about plasmids

Self-replicating, circular DNA molecules. Found in bacteria and some eukaryotes (i.e., yeast, mitochondria, chloroplasts). Bacteria may carry resistance genes for more than one ANTIBIOTIC or ENVIRONMENTAL SUBSTANCE. Our use of antibiotics has lead to the survival and subsequent population increase of bacteria carrying R-factors. R-factors may be transferred between bacteria of the same species, genus, or even different related genera. Commonly used in GENETICALLY ENGINEERING bacteria (i.e., Human Insulin Gene).

What are sense and nonsense codons?

Sense codons: code for amino acids. Nonsense codons: stop codons; do not code for amino acids. Instead the nonsense codons—UAA, UAG, and UGA—signal the end of the protein molecule's synthesis.

Why do prokaryotes not have introns and exons?

The IE hypothesis proposes that introns are very old and were found in both prokaryote and eukaryote ancestors. The idea is that our earliest ancestors had many very small proteins units. Introns then allowed the mixing and matching of these instructions which caused a rapid expansion in the number of genes. Over time, introns were lost from prokaryotes as a way to make proteins more efficiently. This matters in the hurly burly world of bacteria where growing as fast as possible is often critical for survival. In the IL hypothesis, introns appeared after the prokaryotes and eukaryotes went their separate ways. So instead of introns being lost from prokaryotes, they were gained in eukaryotes. Is there an advantage for introns and exons? Remember, most eukaryotes are multicellular organisms. The mixing and matching of exons from the same gene can lead to proteins with different functions. Eukaryotes might need this diversity in proteins because they have many types of cells all with the same set of genes. Therefore, introns are a way to generate different proteins or different amounts of proteins that are unique to a cell type.

When DNA and RNA are copies and transcribed, what is meant by the 5' to 3' direction?

The end with the hydroxyl attached to the 3' carbon is called the 3' end of the DNA strand; the end having a phosphate attached to the 5' carbon is called the 5' end. The way in which the 2 strands fit together dictates that the 5' ---> 3' direction of one strand runs counter to the 5' ---> 3' direction of the other strand.

Describe how a human gene is inserted into bacteria.

The gene is cut from the original DNA source using a restriction enzyme and then pasted into a plasmid by ligation. This is then inserted into a bacterium.

As protein is translated from mRNA, why do they begin with the amino acid methionine?

The start codon that initiates the synthesis of the protein molecule is AUG, which is also the codon for methionine. In bacteria, the start AUG codes for formylmethionine rather than the methionine found in other parts of the protein. The initiating methionine is often removed later, so not all proteins contain methionine.

Do you think transposons might be viruses?

They mimic functions of viral proteins, but can only exist as nucleic acids and have lost the genes that make them true viruses. That is their origin.

What is meant by the promoter site and terminator site of RNA transcription?

Transcription begins when RNA polymerase binds to the DNA at a site called the PROMOTER. Only one of the two DNA strands serves as the template for RNA synthesis for a given gene. Like DNA, RNA is synthesized in the 5' to 3' direction. RNA synthesis continues until RNA polymerase reaches a site on the DNA called the TERMINATOR.

Which type of bacterial recombination involves "naked" DNA and plasmids?

Transformation

Which type of bacterial recombination involves phages (bacterial viruses)?

Transformation by phage

Which type of bacterial recombination is most likely to be used in genetic engineering and why?

Transformation by plasmids

Describe 3 ways that a bacterial gene may be passed to another bacteria.

Transformation, Conjugation, Transposons

What term refers to the process of reading mRNA into protein?

Translation

Which type of bacterial recombination involves "jumping genes"?

Transposons

Which nucleotide is not found in the DNA?

Uracil

What is gel electrophoresis?

a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size

Mutagen

agents that cause mutations

Transduction

bacterial DNA is transferred from a donor cell to a recipient cell inside a virus that infects bacteria, called a BACTERIOPHAGE, or PHAGE. Rare, very sporadic in nature. Once it was commonly used in GENETIC ENGINEERING bacteria.

What does a regulatory gene code for?

called the I gene encodes a REPRESSOR proteins that swtiches inducible and repressible operons ON or OFF.

Why is recombination important?

creates genetic variation among sperm and egg cells

Cloning Strategies

for ISOLATING and COPYING a particular fragment (or GENE) of DNA.

Transformation

genes are transferred from one bacterium to another as "naked" DNA in solution. Example: proved by Griffith in 1928 (though not understood at the time). Bacterial cells after lysis or death release DNA into the environment. Fragments of DNA may be taken up and incorporated by live bacteria through recombination. Works best between RELATED bacteria. **Naturally occurs among Bacillus, Hemophilus, Neisseria, Acinetobacter, and some Streptococcus and Staphylococcus genera. Only in COMPETENT cells (can be forced!) - which can alter CELL WALLS making them more PERMEABLE to DNA fragments. KNOWN AS COMPETENCE!! Through biotechnology, non-competent can be made competent.

What is a GMO? Why could the pose a problem?

genetically modified organism; organisms that have had their characteristics changed through the modification of their DNA. Whether they might causes an allergic reaction or toxicity to human and non-pest species. In the case of genetically modified foods.

What is a triplet and how many amino acids can one code for?

mRNA is made of CODONS, 3 nucleotides code for one amino acid (in DNA it is called a triplet).

What are the 3 types of RNA?

messenger RNA: the type of RNA molecule that directs the incorporation of amino acids into proteins; carries the coded information for making specific proteins from DNA to ribosomes, where proteins are synthesized. transfer RNA: the type of RNA molecule that brings amino acids to the ribosomal site where they are incorporated into proteins; involved in translation; with the ribosomes, recognize the specific codons and transport the required amino acids. ribosomal RNA: forms an integral part of ribosomes, the cellular machinery for protein synthesis.

Spontaneous Mutations

occur in the absence of a mutagen

Vertical Gene Transfer

occurs when genes are passed from an organism to its offspring. Plants and animals transmit their genes by vertical transmission.

What is the function of a ribosome?

protein synthesis or translation

Transposons

small segments of DNA that can move from one region of a DNA molecule to another "jumping genes" They contain insertion sequences for CUTTING and RESEALING DNA (transposase) Barbara McClintock discovered transposons in corn. occur in all organisms! may INACTIVATE if inserted into the middle. But transposition of genes is rare! EXAMPLE: transgenic mice that carry the piggyBAC transposon that has caused their cells to express red fluorescent protein.

RNA that brings the amino acid to the ribosome to build protein:

tRNA

Genetic Recombination

the exchange of GENES between 2 DNA MOLECULES to form new combinations of genes on a chromosome. If a cell picks up a foreign DNA, some of it could insert into the cell's chromosome--a process called CROSSING OVER--and some of the genes carried by the chromosomes are shuffled. In Eukaryotes: occurs only during MEIOSIS. In Prokaryotes: occurs by several methods; more likely than a MUTATION to result in an advantageous combination because WHOLE GENES ARE TRANSFERRED.

Genetics

the study or heredity, structure and function of genes

Genetic Engineering

use of technology to alter the genome of a virus, bacteria, or other cells for medical, agricultural, or industrial purposes.

Cloning

used to produce many identical copies of a particular gene.