Microbiology: Chapter 9: Gene Transfer and Recombination

What are transposons?

- "jumping gene" - a DNA fragment that 'jumps' from one location to another within the chromosome - it carries the gene(s) required for excision from and intersion into DNA

What happens in the Southern Blot technique?

- DNA is separated by electrophoresis - DNA blot on membrane - Labels with specific DNA probe - Detects probe (on X-ray film)

Are any DNA sequences the same? would there be a different cutting pattern by these enzymes passed on a species or individual?

NO! all DNA sequences vary (even within the same species) - the length of a fragment tells information... - the plasmid of known size is cut into pieces that fall into the different benchmarks.

what are the steps of specialized transduction?

Part 1: - begins with a lysogenic cell, in which viral DNA has been inserted into its chromosome - in this case, at phage induction, the viral DNA is cut out of bacterial DNA. however, it is cut improperly so that the viral DNA now has some specific bacterial genes attached to it. - the lytic pathway then follows. the new phage particles are made and the host cell is lysed releasing these Part 2: - Then, one of the phages containing the bacterial DNA, along with the viral DNA, attaches to and injects its DNA into a different host bacterial cell. - The bacteria genes introduced (transduced) into the new cell recombine with the bacterial DNA (possibly along with the viral DNA). Then, bacterial cell could have permanent properties of carrying a toxin or antibiotic resistance.

What are RFLPs?

- Restriction fragment length polymorphisms are different lengths of DNA sequences produced after restriction digests. --> these differ by individual, therefore, we can perform a direct comparison of the DNA of 2 different organisms at a specific site.

how do restriction enzymes recognize where to cut?

- They recognize a known sequence of 4-10 bps at its target so that cutting sites are highly regulated. --> cut at palindromes in DNA that are the same when read in both directions 5'->3' and 3'->5'

what is recombination? what is the end result of it?

- an event where one bacterium donates (or passes) DNA to another bacterial cell. - the end result is usually another strain of bacterium that has the characteristics of the donor cell and the original strain is then called the "recombinant".

What is competence and how does it relate to transformation?

- competence requires cells to be able to take up DNA from the outside of the cell. Some bacterial cells are naturally competent and can take up DNA without an effort. However, some bacterial cells are not naturally competent.... they need to be prepared to be artificially competent.

What is genetic engineering?

- derives useful products and applications that owe their invention to basic research EX: basic science regarding the workings of the electron led to TVs, computers, and cell phones EX: DNA manipulation a.) farmers mate 2 largest pigs for best offspring b.) phenotypes are the product of a particular sequence of DNA-drop of blood=identification.

How are probes used? what generates probes?

- diagnose the cause of an infection from a patient's specimen - identify a culture of unknown bacteria or virus - genotyping (check for certain alleles) RESTRICTION ENZYMES generate PROBES

How do you do the nomenclature of restriction enzymes?

- first letter of bacterial genus - first 2 letters of species - endonuclease # EX: escherichia coli (R strain) = EcoRI EX: Haemophilus influenzae (Type D) = HindIII

What is gel electrophoresis? how does the DNA move across the gel?

- forensic mapping - restriction digests - DNA is negative because of all the phosphate groups, therefore DNA will move toward a positive pole in the gel. - Large sizes at the top and smaller at the bottom. Benchmarks are created by known sizes which are used to compare the unknown DNA size.

transposons can come in what two forms? what can they be used for?

- from pieces of DNA that move within the chromosome of the bacterium - from the chromosome of a bacterium to a plasmid - used to insert antibiotic resistance or other genes into the chromosome of a bacterium from another cell. A plasmid from the other cell which contains the transposon can be transformed into another cell. Then, the transposon can jump out of the plasmid and into the bacterial chromosome of the new cell.

What are examples of DNA sequencing?

- gel electrophoresis: rows with highest length determine the order of nucleotides showing up - histograms: highest markings show the reliability/probability of nucleotide being there

What are characteristics relating to the conjugation and fertility of F plasmid in gram POSITIVE cells?

- have F plasmid - F plasmid only contains genes for: plasmid transfer - Attaches by sticky surface on cell

What are characteristics relating to the conjugation and fertility of F plasmid in gram NEGATIVE cells?

- have F plasmid - F plasmid contains genes for: plasmid transfer and the formation of sex pilus - Attaches by sex pilus on cell

What components compose a proper diagnosis?

- results of examination - culturing the organism (koch's postulates) - biotech assays - blood work

what is transformation? what are bacterial cells that can take up DNA through their cytoplasmic membrane called?

- the process where free-floating small fragments of DNA (from a cell that has been lysed or burst open) or plasmid DNA is taken up by another bacterial cell.

What is conjugation?

- the process where the plasmid DNA is transferred to another cell. - it involves the direct contact between the bacterial cell that is donating the DNA and the bacterial cell that is accepting the DNA.

How are RFLPs used in genetic engineering?

- they are used to grow crops, make kids tall, or use rice to express more vitamins at a cheaper cost! - southern blot - helps to find the gene you are looking for - FISH - fluorescent insite to hybridization - we are adding light to a sequence of DNA on a slide. - sequencing - determines the order of A,T,C, and G's

Why is it beneficial for a bacterial cell to have a restriction endonuclease/restriction enzyme?

- want to chop up bacteriophage DNA to prevent replication of that virus - to prevent the virus from hijacking the host cell's (bacteria) body - can be cut from eukaryotic cells too because all DNA is the same!

What are the genetic engineering steps?

--> in the genomic DNA, the target sequence is where the genetic engineering occurs. CYCLE 1: - Denaturation: heat briefly to separate DNA strands - Annealing: cool to allow primers to form hydrogen bonds with ends of target sequence - Extension: DNA polymerase adds nucleotides to the 3' end of each primer. CYCLE 2: - 4 molecules are made CYCLE 3: - 8 molecules are made and 2 of them match the target sequence.

Specifics of DNA transformation

1. Double-stranded fragment of DNA binding proteins on the outside of the cell. Then, single strand of that DNA fragment moves through the cytoplasmic membrane. the single strand remaining on the outside of the cell will be degraded. Then, DNA on the inside of the cell will recombine with the bacterial DNA chromosome 2. The DNA is going to go to a region on the chromosome that has a similar DNA sequence and trade places with the region - a "homologous region"... this incorporates the DNA into the chromosome. 3. The DNA fragments must incorporated must incorporate themselves into the new bacterial cell genome in order to be replicated 4. New cell trades places with old DNA. Within the cell the old and replaced fragment is degraded by enzymes. The new DNA doesn't match what was replaced. Therefore, a repair mechanism will come in and fix one of the strands to make them both the same. Then, you have either the original cell or the new cell with a new stretch of DNA 5. Now, certain pieces of DNA can be beneficial to obtain by the bacterial cell and those are DNA fragments with antibiotic resistant genes. In this case , the gene would be incorporated into the bacterial DNA. Then, that gene is translated and transcribed, a protein is made to allow the cell to be resistant to antibiotics.

What are the steps of conjugation?

1. F+ cell containing the F plasmid makes its sex pilus by expressing the proteins that make up the sex pilus. 2. Then, the sex pilus of F+ cell comes into contact with the F- cell. 3. Next, the sex pilus brings the two cells close together and creates an opening between the two cells. 4. The F plasmid then begins being transferred to the new cell, while replicating at the same time to leave a copy of the F plasmid in the old cell. 5. Once the entire F plasmid is transferred, the 2 cells separate from each other - both are now F+ cells. --> because it is plasmid DNA, the plasmid can replicate itself and does not have to be incorporated into the bacterial DNA genome to be replicated when cell division occurs.

What are the steps of transformation?

1. although the DNA is double stranded, only single strands will enter the bacterial cell 2. This piece of DNA will enter the cell and recombine with the bacterial chromosome by homologous recombination. 3. Cell will multiply and make new cells with DNA incorporate

What are the 2 forms of transformation and what do they do?

1. electroporation - an electric current can make holes in the cell wall and cytoplasmic membrane of the cell, so that DNA can enter the cell. The holes are temporary and are only present at the time the current is being used in the cells. 2. Heat shock - can temporarily and momentarily shock cells with heat so DNA can enter

What are the requirements of conjugation?

1. fertility plasmid 2. the F (fertility) or sex pilus 3. time

What are the 2 forms of transduction and what happens in them? what genes can be transferred in each form?

1. generalized - the transfer of any gene from the DNA occurs. Fragments of the DNA being donated are packaged into new virus particles that are made at assembly in the lytic bacteriophage infection... ANY gene can be donated in this form. 2. specialized - the transfer of specific genes from the DNA occurs. It occurs after the lysogenic cycle when the prophage is excised from the DNA of the bacterium and the excised region includes bacterial genes as well as nucleic acid. During the lytic cycle, the specific genes are packaged into new virus particles during assembly.

What are the steps of using an enterotube?

1. one tube containing media for 15 biochemical tests is inoculated with an unknown enteris bacterium 2. After incubation, the tube is observed for results 3. The value for each positive test is circled and the number f each group of tests are added to give the ID value 4. Comparing the resultant ID value with the computerized listing shows the organism.

What are the steps in Hfr conjugation?

1. the F+ cell becomes an Hfr cell 2. Sex pilus on the Hfr cell comes in contact with an F- cell 3. the pilus brings the 2 cells close together 4. the transfer of the F plasmid begins. However, the F plasmid is integrated into the bacterial genome. Therefore, the F plasmid begins transfer of the plasmid, but also brings over with it genes from the other bacterial cell's genome --> now the F plasmid is small and can be transferred to a new cell fairly quickly. However, a bacterial genome is huge. Therefore, if transfer with the F plasmid with the bacterial genome is going to occur is will take a long time. 5. Therefore, the transfer begins, but takes too long and not all of the F plasmid or bacterial genome can be transferred. 6. The 2 cells break apart before al lthe DNA is transferred. Now the bacterial cell has genes from the old bacterial cell. However, the F- cell did not get all of the F plasmid (and remains F-) 7. The new DNA is homologously recombined with bacterial genome of the new cell and the bacterial cell now has new genes.

What are the mechanisms of lateral gene transfer in prokaryotes?

1. transformation 2. transduction 3. conjugation

what are the key components of transformation

1. uptake of 'naked' DNA 2. captured by DNA binding proteins 3. competence

Normal Basophil level:

1.5-3% - increased in chicken pox, sinusitis, and diabetes. - histamine and coagulant

Normal Eosinophil level:

10-12% - usually indicates allergies

What are the steps of the Hybridization test?

1a. A solmonella DNA fragment is cloned in E. coli 1b. Cloned DNA fragments are marked with fluorescent dye and separated into single strands - forming DNA probes. 2a. Unknown bacteria are collected on a filter 2b. Cells are lysed and DNA is released 2c. DNA is separated into single strands 3. (BOTH PARTS): DNA probes are added to the DNA from the unknown bacteria 4. DNA probes hybridize with salmonella DNA from sample. Then, excess probe is washed off. Fluorescence indicates presence of salmonella.

Normal Lymphocyte level:

20-40% - raised levels indicate viral infection, cancer, or sometimes autoimmune diseases

Normal Monocyte level:

5-8% - raised indicate viral infection and inflammation - these cells can differentiate into macrophages (b-cells or dendritic cells) - indicate an antigen exists

How do bacterial cells use their ability to cut DNA? How are the enzymes used in researcher's labs?

Bacterial cells cut DNA to protect against the incompatible DNA of bacteriophages or plasmids. - in the lab, enzymes can be used to cleave DNA at desired sites for recombinant DNA technology.

what is the f plasmid?

F plasmid - contains genes necessary to allow for conjugation to take place - has genes that encode the proteins to make the sex pilus- the F plasmids are self-transmissible (can transfer themselves to other cells.

What are other ways (besides biotech assays) to diagnose patients? What do you look for?

Histology and IHC - is the bacteria normal? - is there a loss of filtration or inflammation - look for disease type by staining - do cells contain a specific type of protein Complete blood count and immunophenotype - Normal blood count - If raised, you can see what types of cell are higher and know what that affects/ what it is fighting off by creating more of itself.

What are the steps to general transduction?

Part One: - the bacteriophage attaches to a host bacterial cell and injects its viral DNA into the host cell - the virus comes into the cell and takes over transcription, translation and replication machinery - A nuclease enzyme is made that degrades the bacterial DNA. Then, there are fragments of bacterial DNA floating in the cell and viral DNA - The lytic cycle proceeds from there - with all the parts of new virus particles in assembly to form new particles and release by cell lysis. However, a few virus particles that may contain genes from the bacterium that were accidentally packaged in the new virus particles Part 2: - Now, the phage with the genetic information from the bacterium comes into contact with another cell. - the phage will attach to the cell and inject the DNA into the new bacterial cell. - this will pass the genes from the previous bacterium to the next bacterium - the new bacteria will recombine by homologous recombination with the DNA of a new cell - the new cell will have new genes and be recombinant.

What is the major difference between the PCR and Southern blot techniques?

TIME! - PCR: 96 runs in 3 hours -SB: 1 experiment in 3 days SB exposes person to radioactivity.

What is the process of FISH? what does it do?

Take miotic cells and spread them out.. nucleus is condensed and DNA is tightly wound. when fluorescence is added the markers light up. Chromosomes can be labeled to determine if they are intact.

Why is the radioactive label added to the DNA?

The part that lights up indicates a positive for a specific gene

where do restriction enzymes come from? what do they do?

bacterial cells! - recognize foreign DNA and break the phosphodeister bonds between adjacent nucleotides on both strands of DNA.

How does gene transfer work in conjugation?

chromosomal transfer: - sometimes the F plasmid will integrate into the cell genome. - when this occurs, the F+ cell is now called an Hfr (high frequency recombination) cell.

what are bacterial cells that can take up DNA through their cytoplasmic membrane called?

component cells

how does genetic engineering affect diseases?

dysfunctional genetics cause diseases (in some cases) EX: huntington's disease, insulin-dependent diabetes, adenosine deaminase deficiency, blood clotting.

How does genetic engineering improve the outcome of diseases?

it can actually PREVENT the disease which is unlike any other form of treatment

what does reverse transcriptase do?

it converts RNA to DNA - synthesizes complementary DNA (cDNA) from either messenger, transfer, ribosomal, or a few other types of RNAs. - can synthesize eukaryotic genes from mRNA transcripts without introns (valuable tool for genetic engineering) - viruses can have DNA or RNA that is ds or ss. For RNA viruses, they have packaged reverse transcriptase- takes RNA and puts it in the host. - Needed by RNA viruses because their information can be incorporated into the host genome.

KNOW the process of FISH and how restriction nucleases are important in the process

look at a video.

Do F- cells have an F plasmid?

no!

Whats recombinant DNA technology?

removes genetic material from one organism and combines it with that of a different organism. --> after cutting the DNA with a restriction endonuclease, the DNA must be connected again. LIGASE seals two sticky ends of DNA to form a solid molecule again.

what are gene probes?

short stretches of DNA that will base-pair with a stretch of DNA with a complementary sequence (if one exists in the sample). - If you are performing a PCR looking for hunington's allele, the probe you would use would be the disease causing allele - they Look for a complement with the use of restriction enzymes.

What do restriction enzymes do?

the are enzymes that can clip crosswise at selected positions of DNA

What are restriction fragments?

the pieces of DNA that are produced by restriction endonucleases

What is transduction? what occurs in it?

the process in which DNA is transferred to another bacterial cell by a bacterial virus (bacteriophage). - in transduction, the species of bacteria from which the DNA is being donated and the species of bacteria that is getting the DNA must be the same due to the specificity of bacteriophages for the species of bacteria they infect.

Do F+ cells have an F plasmid?

yes!