MICRO- Chapter: 10,11,12

Identify a few tools used in genetic engineering.

- Polymerase chain reaction (PCR): amplifies the number of copies of DNA and can assist in diagnosis of infections caused by microbes that are difficult to culture. - Recombinant DNA technology - Reverse transcriptase to synthesize cDNA - Mutagens: Create changes in microbes' genomes to change phenotypes Select for and culture cells with beneficial characteristics - Restriction enzymes - Vectors: Nucleic acid molecules that deliver a gene into a cell - Gene libraries: A collection of bacterial or phage clones Each clone in library often contains one gene of an organism's genome - Electroporation: one method used for transfecting cells in cell culture

List the major steps of protein synthesis?

1. Dna double helix unwinds 2. mRNA transcribed 3. mRNA leaves nucleus 4. mRNA binds to ribosome 5. tRNA molecules pick up amino acids 6. tRNA anticodon links with mRNA codon 7. Ribosome bonds amino acids together 8. Polypeptide chain completed

DNA (Deoxyribonucleic Acid)

A complex molecule containing the genetic information that makes up the chromosomes. Was discovered and characterized long before its role in heredity was understood. Microbiologists played significant roles in demonstrating that DNA is the hereditary information found within cells.

Explain the impact or effect of point mutations versus frameshift mutations.

A point mutation affects a single base pair such as a silent mutation, missense mutation or nonsense mutation. Whereas frameshift mutation results from an insertion or deletion of a number of nucleotides that is not a multiple of three. The change in reading frame alters every amino acid after the point of the mutation and results in a nonfunctional protein.

true

A single change in a single base is called a frameshift mutation true or false?

Exon-encoded RNA

An exon is any part of a gene that will encode a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term exon refers to both the DNA sequence within a gene and to the corresponding sequence in RNA transcripts.

silent mutations.

As a consequence of the degeneracy of the genetic code, a point mutation will commonly result in the same amino acid being incorporated into the resulting polypeptide despite the sequence change. This change would have no effect on the protein's structure

Single-stranded binding proteins (Identify the major enzymes used during DNA replication.)

Bind to single-stranded DNA to prevent hydrogen bonding between DNA strands, reforming double-stranded DNA

Meiosis

Cell division that produces reproductive cells in sexually reproducing organisms

Explain the 2 functions of the genome.

DNA is the heritable material passed from parent to offspring. DNA directs and regulates the necessary proteins.

Genetic engineering

Direct alteration of an organism's genetics to achieve desirable traits

Explain how RNA is synthesized from DNA.

During the process of transcription, the information encoded within the DNA sequence of one or more genes is transcribed into a strand of RNA, also called an RNA transcript. The resulting single-stranded RNA molecule, composed of ribonucleotides containing the bases adenine (A), cytosine (C), guanine (G), and uracil (U), acts as a mobile molecular copy of the original DNA sequence. Transcription in prokaryotes and in eukaryotes requires the DNA double helix to partially unwind in the region of RNA synthesis. The unwound region is called a transcription bubble. Transcription of a particular gene always proceeds from one of the two DNA strands that acts as a template, the so-called antisense strand. The RNA product is complementary to the template strand of DNA and is almost identical to the nontemplate DNA strand, or the sense strand. The only difference is that in RNA, all of the T nucleotides are replaced with U nucleotides; during RNA synthesis, U is incorporated when there is an A in the complementary antisense strand.

DNA Polymerase I (Identify the major enzymes used during DNA replication.)

Exonuclease activity removes RNA primer and replaces it with newly synthesized DNA

mutagen.

Exposure to mutagens can cause induced mutations, which are various types of chemical agents or radiation. Exposure to a mutagen can increase the rate of mutation more than 1000-fold. Mutagens are often also carcinogens, agents that cause cancer. However, whereas nearly all carcinogens are mutagenic, not all mutagens are necessarily carcinogens.

Briefly describe how DNA, RNA, and/or proteins can be visualized and analyzed.

Finding a gene of interest within a sample requires the use of a single-stranded DNA probe labeled with a molecular beacon typically radioactivity or fluorescence that can hybridize with a complementary single-stranded nucleic acid in the sample.

Eukaryotes (Identify differences between DNA replication in bacteria versus prokaryotes.)

Genome structure: Multiple linear chromosomes Number of origins per chromosome: Multiple Rate of replication: 100 nucleotides per second Telomerase: Present RNA primer removal: RNase H Strand elongation: pol δ, pol ε

Prokaryotes (Identify differences between DNA replication in bacteria versus prokaryotes).

Genome structure: Single circular chromosome Number of origins per chromosome: Single Rate of replication: 1000 nucleotides per second Telomerase: Not present RNA primer removal: DNA pol I Strand elongation: DNA pol III

Sliding clamp (Identify the major enzymes used during DNA replication.)

Helps hold DNA pol III in place when nucleotides are being added

The plasmids may contain the genes for making an antibiotic, bacteriocin, fertility factor, etc.

How may plasmids help cells survive?

What is happening during translation?

In translation, polypeptides are synthesized using mRNA sequences and cellular machinery, including tRNAs that match mRNA codons to specific amino acids and ribosomes composed of RNA and proteins that catalyze the reaction.

Topoisomerase IV (Identify the major enzymes used during DNA replication.)

Introduces single-stranded break into concatenated chromosomes to release them from each other, and then reseals the DNA

The genotype of a cell is the full collection of genes a cell contains. Not all genes are used to make proteins simultaneously. The phenotype is a cell's observable characteristics resulting from the proteins it is producing at a given time under specific environmental conditions.

Is there a difference between genotype and phenotype? What are they?

Transformation: mechanism of horizontal gene transfer in which naked environmental DNA is taken up by a bacterial cell. Transduction: Mechanism of horizontal gene transfer in bacteria in which genes are transferred through viral infection Conjugation: Transfer of DNA through direct contact using conjugation pilus.

List and briefly explain the 3 processes that describe how prokaryotes can gain or exchange genetic information to become more diverse.

DNA polymerase III (Identify the major enzymes used during DNA replication.)

Main enzyme that adds nucleotides in the 5' to 3' direction

Miescher, Swiss (1844-1895): physician who isolated DNA from white blood cells from pus on used bandages that he called: nuclein. Richard Altmann, student of Miescher called it Nucleic acid Joachim Hammerling (1901-1980): The cells of the single-celled alga Acetabularia measure 2-6 cm and have a cell morphology that can be observed with the naked eye. Each cell has a cap, a stalk, and a foot, which contains the nucleus. Hämmerling found that if he removed the cap, a new cap would regenerate; but if he removed the foot, a new foot would not regenerate. He concluded that the genetic information needed for regeneration was found in the nucleus

Many significant experiments were mentioned. Identify 2 historical figures who advanced the knowledge of DNA or inheritance and briefly explain their contribution(s).

Transduction

Mechanism of horizontal gene transfer in bacteria in which genes are transferred through viral infection

False

Mitosis results in daughter cells containing half the number of chromosomes as compared to the parent cell? true or false?

Eukaryotes (Compare and contrast transcription in prokaryotes versus eukaryotes.)

Number of polypeptides encoded per mRNA: Exclusively monocistronic Strand elongation: RNA polymerases I,II, or III Addition of 5' cap: Yes Addition of 3' poly-A-tail: Yes Splicing of pre-mRNA: Yes

Prokaryotes (Compare and contrast transcription in prokaryotes versus eukaryotes.)

Number of polypeptides encoded per mRNA: Monocistronic or polycistronic Strand elongation: core + σ = holoenzyme Addition of 5' cap: No Addition of 3' cap: No Splicing of pre-mRNA: No

Explain how the genetic code is degenerate.

Of these 64 codons, 61 represent amino acids, and three are stop signals. Although each codon is specific for only one amino acid (or one stop signal), the genetic code is described as degenerate, or redundant, because a single amino acid may be coded for more than one codon.

Helicase (Identify the major enzymes used during DNA replication.)

Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases

Define missense mutations.

Point mutation that results in a different amino acid being incorporated into the resulting polypeptide. The effect of a missense mutation depends on how chemically different the new amino acid is from the wild-type amino acid. The location of the changed amino acid within the protein also is important. For example, if the changed amino acid is part of the enzyme's active site, then the effect of the missense mutation may be significant. Many missense mutations result in proteins that are still functional, at least to some degree. Sometimes the effects of missense mutations may be only apparent under certain environmental conditions; such missense mutations are called conditional mutations. Rarely, a missense mutation may be beneficial. Under the right environmental conditions, this type of mutation may give the organism that harbors it a selective advantage.

transformation

Raw (foreign) DNA may be accepted by another similar cell in a process called

Topoisomerase II (DNA gyrase) (Identify the major enzymes used during DNA replication.)

Relaxes supercoiled chromosome to make DNA more accessible for the initiation of replication; helps relieve the stress on DNA when unwinding, by causing breaks and then resealing the DNA

Describe the structure of RNA.

Ribonucleotides are typically single stranded and contain the pentose sugar ribose instead of the deoxyribose found in DNA. RNA contains the pyrimidine uracil in place of thymine found in DNA. Although RNA is not used for long-term genetic information in cells, many viruses do use RNA as their genetic material. There are 3 main types of RNA, all involved in protein synthesis: Messenger RNA (mRNA): serves as the intermediary between DNA and the synthesis of protein products during translation. Ribosomal RNA (rRNA): is a type of stable RNA that is a major constituent of ribosomes. It ensures the proper alignment of the mRNA and the ribosomes during protein synthesis and catalyzes the formation of the peptide bonds between two aligned amino acids during protein synthesis. Transfer RNA (tRNA): is a small type of stable RNA that carries an amino acid to the corresponding site of protein synthesis in the ribosome. It is the base pairing between the tRNA and mRNA that allows for the correct amino acid to be inserted in the polypeptide chain being synthesized.

Ligase (Identify the major enzymes used during DNA replication.)

Seals the gaps between the Okazaki fragments on the lagging strand to create one continuous DNA strand

Summarize the mechanisms, risks, and potential benefits of gene therapy.

So far, gene therapies have proven relatively ineffective, with the possible exceptions of treatments for cystic fibrosis and adenosine deaminase deficiency, a type of SCID. Other trials have shown the clear hazards of attempting genetic manipulation in complex multicellular organisms like humans. In some patients, the use of an adenovirus vector can trigger an unanticipated inflammatory response from the immune system, which may lead to organ failure. Moreover, because viruses can often target multiple cell types, the virus vector may infect cells not targeted for the therapy, damaging these other cells and possibly leading to illnesses such as cancer. Another potential risk is that the modified virus could revert to being infectious and cause disease in the patient. Lastly, there is a risk that the inserted gene could unintentionally inactivate another important gene in the patient's genome, disrupting normal cell cycling and possibly leading to tumor formation and cancer. Because gene therapy involves so many risks, candidates for gene therapy need to be fully informed of these risks before providing informed consent to undergo the therapy.

Explain the difference between somatic cell and germ-line gene therapy.

Somatic cells are non-reproductive cells within the body, because somatic cell traits are not inherited any genetic changes accomplished by somatic-cell gene therapy would not be passed on to offspring. However, should scientists successfully introduce new genes to germ cells (eggs or sperm), the resulting traits could be passed on to offspring. This approach, called germ-line gene therapy, could potentially be used to combat heritable diseases, but it could also lead to unintended consequences for future generations.

Primase (Identify the major enzymes used during DNA replication.)

Synthesizes RNA primers needed to start replication

Epigenetics

The influence of environmental factors on DNA packaging is called epigenetics. Epigenetics is another mechanism for regulating gene expression without altering the sequence of nucleotides. Epigenetic changes can be maintained through multiple rounds of cell division and, therefore, can be heritable.

Recombinant DNA

The process by which a DNA sequence is manipulated in vitro, thus creating recombinant DNA molecules that have new combinations of genetic material

Biotechnology

The science of using living systems to benefit humankind

Intron-encoded RNA

The term intron refers to both the DNA sequence within a gene and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final mature RNA after RNA splicing are exons.

Semiconservative replication is used to describe how DNA is copied, what does this mean?

There were three models suggested for DNA replication. In the conservative model, parental DNA strands (blue) remained associated in one DNA molecule while new daughter strands (red) remained associated in newly formed DNA molecules. In the semiconservative model, parental strands separated and directed the synthesis of a daughter strand, with each resulting DNA molecule being a hybrid of a parental strand and a daughter strand. In the dispersive model, all resulting DNA strands have regions of double-stranded parental DNA and regions of double-stranded daughter DNA.

Barbara Mcclintock

This scientist discovered "jumping genes"

Conjugation

Transfer of DNA through direct contact using conjugation pilus.

What does bidirectional DNA replication mean and why does this occur?

Two replication forks are formed at the origin of replication, allowing for bidirectional replication and formation of a structure that looks like a bubble when viewed with a transmission electron microscope; as a result, this structure is called a replication bubble. The DNA near each replication fork is coated with single-stranded binding proteins to prevent the single-stranded DNA from rewinding into a double helix.

Describe why regulation of operons is important.

Understanding the interaction between the gene expression of a pathogen and that of its human host is important for the understanding of a particular infectious disease. Gene regulation involves a complex web of interactions within a given cell among signals from the cells environment, signaling molecules within the cell, and the cell's DNA. These interactions lead to the expression of some genes and the suppression of others, depending on circumstances. In single-celled organisms like prokaryotes, it primarily ensures that a cell's resources are not wasted making proteins that the cell does not need at that time.

Describe the structure of DNA.

Watson and Crick proposed the double helix model for DNA. Each DNA is made up of a sugar called, deoxyribose, a phosphate group, and a nitrogenous base in this case, adenine. DNA contains the pyrimidines cytosine and thymine, and the purines adenine and guanine. The two DNA strands are antiparallel to each other. The direction of each strand is identified by numbering the carbons (1 through 5) in each sugar molecule. The 5' end is the one where carbon #5 is not bound to another nucleotide; the 3' end is the one where carbon #3 is not bound to another nucleotide. Hydrogen bonds form between complimentary nitrogenous bases on the interior of DNA. In the laboratory, the double helix can be denatured to single-stranded DNA through exposure to heat or chemicals, and then renatured through cooling or removal of chemical denaturants to allow the DNA strands to reanneal.

DNA is usually double stranded and most cellular RNA is single stranded. In DNA the pentose sugar is deoxyribose. Whereas in RNA the pentose sugar is ribose. In DNA the common organic bases are adenine,guanine,cytosine and thymine. In RNA the common organic bases are adenine,guanine,cytosine and uracil.

What are at least 3 differences between RNA & DNA?

You are able to achieve desirable traits, modify foods, produce products such as insulin, vaccines, and biodegradable polymers.

What are some benefits of genetic engineering?

cDNA

When mRNA is used to make DNA

methionine

Which is the amino acid that appears at the N-terminus of all newly translated prokaryotic and eukaryotic polypeptides

Hershey and Chase

Which scientists worked with bacteriophages to show DNA is injected into the host cell?

Watson and Crick.

Which two people are given the credit for using clues to construct the widely accepted double stranded model of the DNA structure?

Genotype or genome

a cell remains CONSTANT whereas its phenotype changes in response to environmental influences

nucleoside analog

a chemical mutagen that is structurally similar to a nucleotide but has different base paring rules

Reporter gene

a gene whose expression can be easily visualized and monitored

Polycistronic

a mRNA is one that codes for multiple polypeptides

transpoon or transposable element

a small DNA molecule that has the ability to independently excise from one location in a larger DNA molecule and integrate into the DNA elsewhere

Genome

all the genetic information of the organism

Restriction endonucleases (restriction enzymes

bacterial enzymes produced as a protection mechanism to cut and destroy foreign cytoplasmic DNA that is most commonly a result of bacteriophage infection. Categorized into two groups based on type of cut such as cuts with sticky ends or cuts with blunt ends.

Mitosis

cell division in which the nucleus divides into nuclei containing the same number of chromosomes

Define nonsense mutations.

converts a codon encoding an amino acid (a sense codon) into a stop codon (a nonsense codon). Nonsense mutations result in the synthesis of proteins that are shorter than the wild type and typically not functional.

Chromosome

discreet DNA structures with RNA and associated proteins

Plasmid

extrachromosomal DNA in prokaryotes is commonly maintained that encode a few nonessential genes that may be helpful under specific conditions. Plasmids can be spread through a bacterial community by horizontal gene transfer.

Genotype

full collection of genes that a cell contains within its genome.

Chromosomal theory of inheritance

in 1902, Theodor Boveri observed that in sea urchins, nuclear components (Chromosomes) determined proper embryonic development. That same year, Walter Sutton observed the separation of chromosomes into daughter cells during meiosis. Together, these observations led to the development of the theory, which identified chromosomes as the genetic material responsible for Mendelian inheritance.

False

in agarose gel electrophoresis, DNA will be attracted to the negative electrode

Endonucleases or restriction enzymes

in the DNA code, sequences read the same left to right and right to left that are recognized by BLANK to cut the DNA

Horizontal gene transfer

is a group of mechanisms that allow for the introduction of genetic material from one organism to another organism within the same generation

Transfer RNA (tRNA) (There are 3 main types of RNA, all involved in protein synthesis)

is a small type of stable RNA that carries an amino acid to the corresponding site of protein synthesis in the ribosome. It is the base pairing between the tRNA and mRNA that allows for the correct amino acid to be inserted in the polypeptide chain being synthesized.

Ribosomal RNA (rRNA) (There are 3 main types of RNA, all involved in protein synthesis)

is a type of stable RNA that is a major constituent of ribosomes. It ensures the proper alignment of the mRNA and the ribosomes during protein synthesis and catalyzes the formation of the peptide bonds between two aligned amino acids during protein synthesis

Phenotype

is the observable characteristics of a cell (or organism) at a given point in time and results from the complement of genes currently being used.

Inducible operons

like the lac operon of E. coli, often contain genes encoding enzymes in a pathway involved in the metabolism of a specific substrate like lactose. These enzymes are only required when that substrate is available, thus expression of the operons is typically induced only in the presence of the substrate.

Repressible operons

like the tryptophan (trp) operon, typically contain genes encoding enzymes required for a biosynthetic pathway. As long as the product of the pathway, like tryptophan, continues to be required by the cell, a repressible operon will continue to be expressed. However, when the product of the biosynthetic pathway begins to accumulate in the cell, removing the need for the cell to continue to make more, the expression of the operon is repressed.

Transformation

mechanism of horizontal gene transfer in which naked environmental DNA is taken up by a bacterial cell.

Nucleotide bases

nitrogen bases, cytosine, thymine, guanine, adenine

Messenger RNA (mRNA) (There are 3 main types of RNA, all involved in protein synthesis)

serves as the intermediary between DNA and the synthesis of protein products during translation.

What are Okazaki fragments?

short fragment of DNA made during lagging strand synthesis

What is the central dogma of molecular biology? What is the flow of the information?

states that DNA encodes messenger RNA, which in turn, encodes protein. Each of the processes of replications, transcription, and translation includes the stages of 1) initiation 2) elongation (polymerization and 3) termination.

Operator

the DNA sequence to which repressors may bind, that lies b/w the promoter and the first structural gene

Dna gyrase or topisomerase

the enzyme responsible for relaxing supercoiled DNA to allow for the initiation of replication

aminoacyl-tRNA synthetase

the enzyme that adds an amino acid to a tRNA molecule

photolyase

the enzyme used in light repair to split thymine dimers

Pharmacogenomics (toxicogenomics

the evaluation of the effectiveness and safety of drugs on the basis of information from an individual's genomic sequence as well as examination of changes in gene expression in response to the drug

Wild Type

the phenotype of an organism that is most commonly observed in nature

Catabolite repression

the prevention of expression of operons encoding substrate use pathways for substrates other than glucose when glucose is present

Transcription

the process of making an RNA copy of a gene

Spliceosome

the protein complex responsible for removing intron-encoded RNA sequences from primary transcripts in eukaryotes

metagenomics

the science of studying genomic fragments from microbial communities allowing researchers to study genes from a collection of multiple species

transcriptomics

the science of studying the entire collection of mRNA molecules produced by cells, allowing scientists to monitor differences in gene expression patterns b/w cells

Genomic

the study and comparison of entire genomes, including the complete set of genes, their nucleotide sequence and organization, and their interactions within a species and with other species

wobble position

the third position within a codon, in whcih changes often result in the incorporation of the same amino acid into the growing polypeptide

Inheritance

transmission of traits from one generation to another

Rolling circle replication

unidirectional replication of a circular DNA molecule like a plasmid that involves nicking one DNA strand and displacing it while synthesizing a new strand

lactose present; glucose absent

what leads to the maximum expression of the lac operon?

One gene-one enzyme hypothesis

which suggested that each gene encodes one enzyme.

Polyacrylamide gel electrophoresis

which technique is used to separate proteins fragments based on SIZE

RFLP

which technique uses restriction enzyme digestion followed by agarose gel electrophoresis to generate a handing pattern for comparison to another sample processed in the same way