DNA sequencing of the entire H. influenzae genome was...

DNA sequencing of the entire $H$. influenzae genome was completed in $1995 .$ When DNA from the nonpathogenic strain $H$. influenzae $R d$ was compared to that of the pathogenic $b$ strain, eight genes of the fimbrial gene cluster (located between the $p u r E$ and pep$N$ genes involved in adhesion of bacteria to host cells were completely missing from the nonpathogenic strain. What effect would this deletion have on cotransformation of $p u r E$ and $p e p N$ genes using DNA isolated from the nonpathogenic versus the pathogenic strain? Genes encoding toxins are often located on plasmids. A recent outbreak has just occurred in which a bacterium that is usually nonpathogenic is producing a toxin. Plasmid DNA can be isolated from this newly pathogenic bacterial strain and separated from the chromosomal DNA. To establish whether the plasmid DNA contains a gene encoding the toxin, you could determine the sequence of the entire plasmid and search for a sequence that looks like other toxin genes previously identified. An easier way exists to determine whether the plasmid DNA carries the gene(s) for the toxin; this strategy does not involve DNA sequence analysis. Describe this easier method.

Key Concepts

Cotransformation and Linkage Mapping

Cotransformation refers to the process by which two or more genetic markers located sufficiently close together on a chromosome are simultaneously taken up during transformation. The frequency of cotransformation is influenced by the physical distance between the markers; a smaller distance generally increases the probability that both markers will be incorporated into the recipient genome. In the context of this question, deletion of intervening genes reduces the physical gap between two flanking markers, therefore potentially increasing their cotransformation frequency.

Plasmid-mediated Gene Transfer and Phenotypic Screening

Many bacterial toxins are encoded on plasmids, which are small, independent DNA molecules that can be transferred between cells. An easier method to determine whether a plasmid carries a toxin gene—without performing complete DNA sequencing—is to introduce the isolated plasmid into a recipient strain that normally does not produce the toxin, using a transformation assay. If the recipient expresses the toxin phenotype after acquiring the plasmid, it indicates that the toxin gene is present on that plasmid.

Transcript

00:01 Hi, everyone.

00:02 My name is eric, and let's review problem 47.

00:05 So in this question, they are asking us to describe the process of molecular cloning.

00:13 Now, molecular cloning is the process where you take your dna of interest, you insert it in a plasmid, and then you're going to place it in a host to replicate and produce more amounts of your dna of interest.

00:26 And the process to do that is actually what we're looking for here.

00:31 So we're looking at four different choices, a, b, c, and d.

00:35 And i'll be walking us through what is the correct answer and why the other answers are not correct.

00:41 So the first answer is letter a.

00:45 So let's start with a.

00:46 So they're saying that the foreign dna and the plasmid are cut with the same restriction enzyme and the dna is inserted into a gene.

00:55 So let's start there.

00:55 So our plasmid is a circular piece of dna, and it's mostly common found in the bacteria.

01:05 So with the plasmid, we are going to cut at specific sites with a restriction enzyme, and that's going to allow us to insert our foreign dna into the same spot.

01:18 And in the problem, they described it as cutting into the lack z gene.

01:25 So by cutting into the laxc -z gene, we are actually going to be disrupting this gene.

01:32 And the lax -z gene is responsible for metabolizing lactose.

01:40 So lactose is a molecule, and lax -z is the gene that allows the host to break down and utilize lactose.

01:50 So that would be the first part where we're going to be inserting our dna of interest inside the lactose.

01:57 Lachy gene, right? and then they said that the dna and the vector are annealing.

02:03 So then that would allow us to make a new plasmid, but instead our dna of interest will be bonded in the spot where the llaxi should be.

02:15 And then this plasmid is going to be transferred into a bacterial host, and it's going to be selected for using ampicillin.

02:26 Right.

02:26 So somewhere on this plasmid, will be an amp resistant gene, so that allows the bacteria to survive on ampicillin.

02:36 So that's how we know if the plasmid has been taken up into the bacteria.

02:41 The ones that survive on amp resistance is what we're looking for.

02:46 And once we have it into a bacterial host, we're going to be able to produce more plasmids with our dna of interest...

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