2002-03-14T23:39:00.000-08:00

GENE CLONING – T.A. BROWN

PART ONE – THE BASIC PRINCIPLES OF GENE CLONING

CH.1 - INTRO

Basic steps of a gene cloning experiment:

1. A fragment of DNA, containing the gene to be cloned is inserted into a VECTOR (circular DNA molecule capable of replication when inserted into the host organism.) When these two are joined they form a CHIMERA or RECOMBINANT DNA MOLECULE.

2. The vector(VEHICLE) is inserted into the living host cell (usually a bacterium, though not always.)

3. The vector multiplies within the host cell.

4. When the host cell divides, copies of the vector are passed to the progeny and further replication continues.

5. After many cell divisions, a colony of cloned host cells is produced, each of which will contain one or more copies of the recombinant DNA molecule (vector).

CH.2 - VEHICLES

The vehicle transports the gene into the host cell and is responsible for it’s replication.
A good vehicle must possess the following characteristics:

1. Able to replicate within the host cell
2. Relatively small. Ideally less than 10 kilobases (kb), as large molecules tend to break down in purification, and are also difficult to manipulate.

Two naturally occurring types of DNA molecule that can do this are:

1. PLASMIDS – Small circles of DNA found in bacteria and some other organisms. Able to replicate independently of the host cell chromosome.

2. VIRUS CHROMOSOMES – in particular the chromosomes of BACTERIOPHAGES, which are viruses that infect bacteria.

PLASMIDS

- Circular DNA molecules that lead an independent existance in the bacterial cell.
- Almost always carry one or more genes, which are often responsible for a useful characteristic displayed by the host bacterium Eg: Antibiotic resistance of a bacterium is usually due a antibiotic-resistance gene within a plasmid in the bacterial cell.
- Such antibiotic resistance is often used as a SELECTABLE MARKER in the lab to ensure that bacteria in a culture contain a particular plasmid.
- All plasmids possess at least one DNA sequence that can act as an ORIGIN OF REPLICATION, so they are able to multiply independently of the main bacterial chromosome.
- Smaller plasmids do this by making use of the host cell’s own replicative enzymes. Some larger plasmids carry genes that code for special enzymes which are specifically for plasmid replication. (Q:Do these enzymes interact with/affect the host cell or it’s chromosome/enzymes??)
- A few types of plasmids replicate by inserting themselves into the bacterial chromosome, stably maintained in this form through numerous cell divisions, but will, at some stage, exist as independent elements once again. (Q:How do they insert themselves….how do they know when to separate again….divine intervention!?!) These are called EPISOMES or INTEGRATIVE PLASMIDS….(The others being NON-INTEGRATIVE PLASMIDS)

SIZE AND COPY NUMBER

- Both very important as far as cloning is concerned.
- Plasmid size varies from 1 kb to over 250 kb, so only a few are useful for cloning ie:<10kb
- Larger plasmids can be adapted for cloning under some circumstances…details later in book.
- COPY NUMBER refers to the number of molecules of a plasmid in a single bacterial cell.
- The factors that control copy no. are NOT WELL UNDERSTOOD, but each plasmid has a characteristic value that can be as low as 1 (especially for large molecules) or as many as 50 or more.
- A useful cloning vehicle needs to be present in the cell in multiple copies so that large quantities of the recombinant DNA molecule can be obtained.

CONJUGATION AND COMPATIBILITY

- Plasmids fall into two categories (Q:I take it this should be “non-integrative plasmids…” as he’s already divided them into ‘Integrative’ and ‘Non-Integrative’…clarify…)
- After that little ramble, those two categories are CONJUGATIVE and NON-CONJUGATIVE.
- CONJUGATIVE plasmids promote sexual conjugation between bacterial cells (“And ye the lord said ‘Go forth and multiply’…and thus there was much bacterial frivolity…”)
- Actually…this doesn’t make the cells multiply, rather, the plasmid convinces the cell that it should pass on the plasmid’s DNA to another cell. Whether this is done by some smooth talking, or a little bribery is not known, but most of the whitecoat brigade seem to think that two cells connect via a PHYLUS (hollow appendage present on the surface of the donor cell) and then a copy of the plasmid is passed through to the recipient cell. Another possibility is that it is transferred directly across the bacterial cell wall. A very base interaction and they have NO IDEA….another good place to come back to later…
- Conjugations and plasmid transfer are controlled by a set of ‘transfer’ genes or tra genes, which are obviously present on conjugative plasmids, but not on non-conjugatives.
- Non-conjugative plasmids can be transferred in this way sometimes by hitching a ride if a conjugative plasmid is present in the cell.
- Several kinds of plasmid can be found in the same cell, providing they are COMPATIBLE
- If two incompatible plasmids are present, one or the other will be quite rapidly lost from the cell…(HOW???Chemical reaction? Like or unlike charges?)
- Different types of plasmid can be assigned to different INCOMPATIBILTY GROUPS on the basis of whether or not they can coexist.
- Plasmids from a single incompatibility group are often related to each other in various ways.(How?)
- The basis of incompatibility is NOT WELL UNDERSTOOD, but events during replication are thought to underlie the phenomenon.

PLASMID CLASSIFICATION

2002-03-14T23:38:00.000-08:00

Cloning