Biology Chapter 5: DNA Introduction in Cells

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Questions and Answers

What is the main purpose of cloning in gene cloning experiments?

To enhance the quality of the initial DNA sample

To produce large amounts of recombinant DNA from a limited amount (correct)

To control the growth of bacterial cells

To increase the variety of DNA sequences available

Which statement correctly describes how cloning aids in purification?

It often results in other DNA molecules being present (correct)

It decreases the purity of recombinant DNA

It ensures that only one type of DNA molecule is present

It can be controlled to eliminate all unwanted DNA

In bacterial cloning, how much increase in DNA can typically be obtained from a single bacterial colony?

A hundredfold increase

A tenfold increase

A ten-thousandfold increase

A thousandfold increase (correct)

What does the term 'cloning' strictly refer to in the context of gene cloning?

The production of colonies of cells containing recombinant DNA Signup and view all the answers

What can be directly inferred about the yield of DNA from cultured cells compared to initial amounts?

Cultured cells can yield a millionfold increase in DNA Signup and view all the answers

Which process is primarily involved in the uptake of DNA by bacterial cells?

Transformation Signup and view all the answers

What happens to a bacterium that takes up a plasmid during cloning?

It divides to produce multiple copies of the plasmid Signup and view all the answers

Why is cloning important for molecular biological studies?

It provides large amounts of DNA needed for these studies Signup and view all the answers

What is the primary method used to introduce DNA into animal cells through a liquid medium?

Liposome fusion Signup and view all the answers

In the context of plant cell transformation, what is the role of the protoplast?

To facilitate DNA entry through the cell wall Signup and view all the answers

Which step follows the degradation of the cell wall during the transformation of plant protoplasts?

Cell wall reforming Signup and view all the answers

What is a significant advantage of using liposomes for DNA delivery into animal cells?

It is a non-viral method of gene transfer. Signup and view all the answers

What is one disadvantage of precipitating DNA onto animal cells?

It is less efficient than liposome methods. Signup and view all the answers

What occurs during the fusion of DNA-containing liposomes with animal cells?

Liposomes merge with the cell membrane to release DNA. Signup and view all the answers

Which genus of bacteria is noted for being easily transformable?

Bacillus Signup and view all the answers

What is a key characteristic of competent bacterial cells?

They have enhanced ability to take up DNA. Signup and view all the answers

What is the outcome after the transformation of plant protoplasts?

The regeneration of transformed plant cells. Signup and view all the answers

What treatment is commonly used to prepare competent E. coli cells?

Soaking in ice cold salt solution. Signup and view all the answers

Which method involves the direct insertion of DNA into a cell using a fine needle?

Microinjection Signup and view all the answers

Which of the following salts is traditionally used to prepare competent cells?

Calcium chloride. Signup and view all the answers

Why do most species of bacteria take up only limited amounts of DNA under normal circumstances?

They lack sophisticated mechanisms for uptake. Signup and view all the answers

What effect does calcium chloride (CaCl2) have on competent cells?

It helps in the precipitation of DNA onto the cells. Signup and view all the answers

How does the transformation process in bacteria typically occur in nature?

It is not a major process for genetic information acquisition. Signup and view all the answers

What happens to E. coli cells during the treatment with 50 mM CaCl2?

They become more competent for DNA uptake. Signup and view all the answers

What is the main purpose of plating onto a selective medium?

To distinguish transformants from non-transformants Signup and view all the answers

What happens to the gene product when new DNA is inserted into the vector?

The gene product is disrupted Signup and view all the answers

What does insertional inactivation refer to?

The disruption of a gene leading to a missing product Signup and view all the answers

What characteristic do transformed colonies typically display compared to non-transformants?

Presence of selectable or identifiable traits Signup and view all the answers

Which of the following best describes the molecular structure of the plasmid pBR322?

It features multiple resistance genes like ampR and tetR Signup and view all the answers

What is a common method used to insert foreign DNA into a plasmid vector?

Insertion utilizing enzymatic digestion and ligation Signup and view all the answers

Which enzyme is typically used to cut the plasmid at a specific site for foreign DNA insertion?

Restriction endonuclease Signup and view all the answers

Which factor is crucial for differentiating between recombinant and self-ligated vector molecules?

The presence of selectable markers which are expressed Signup and view all the answers

What occurs when a DNA fragment is inserted into pBR322?

One of the genes present on the plasmid is inactivated. Signup and view all the answers

What characterizes recombinants formed with pBR322?

They display sensitivity to tetracycline. Signup and view all the answers

What is the antibiotic resistance profile of cells containing a recombinant pBR322 molecule?

Ampicillin resistant and tetracycline sensitive. Signup and view all the answers

Which restriction enzyme cuts pBR322 at a location related to the tetracycline resistance gene?

BamHI Signup and view all the answers

How can pBR322 recombinants be screened for successfully integrated DNA fragments?

By assessing their growth on tetracycline medium. Signup and view all the answers

What happens to the tetracycline resistance gene after the insertion of new DNA in the BamHI site of pBR322?

The gene is inactivated. Signup and view all the answers

Why might cells carrying recombinant pBR322 still grow on ampicillin medium?

The ampicillin resistance gene remains intact. Signup and view all the answers

What kind of medium would typically not support the growth of recombinants with pBR322?

Agar containing tetracycline. Signup and view all the answers

What are the components that may be present in a ligation mixture?

Unligated vector molecules and mismatched recombinant DNA Signup and view all the answers

Why are unligated molecules generally not problematic in a ligation mixture?

They are often degraded by the host bacteria's enzymes. Signup and view all the answers

What is a critical challenge in cloning involving recombinant DNA?

Distinguishing correct recombinant DNA from incorrect ones Signup and view all the answers

What happens to most DNA molecules taken up by bacteria from their environment?

They are often degraded within the host cell. Signup and view all the answers

How does cloning allow for the purification of desired DNA molecules despite the presence of other types?

Only one DNA molecule can be taken up per cell. Signup and view all the answers

What role do self-ligated vector molecules play in bacterial transformation?

They replicate just as efficiently as the desired recombinant molecules. Signup and view all the answers

What is the primary function of the chapter regarding transformation?

To describe the introduction of vectors and recombinant molecules into bacterial cells. Signup and view all the answers

When cloning, how are different colonies typically characterized?

Each colony represents different DNA molecules. Signup and view all the answers

Study Notes

Chapter 5: Introduction of DNA into Living Cells

Recombinant DNA molecules are created using methods described in Chapter 4.
The next step in gene cloning involves introducing these molecules into living cells (usually bacteria) to create clones.
Cloning serves two main purposes:
- Producing a large number of recombinant DNA molecules from small amounts of starting material. Bacteria taking up plasmids multiply to form large colonies, and each cell in the colony contains multiple copies of the DNA.
- Purifying recombinant DNA molecules. Cloning helps minimize other DNA molecules being present in the final product.
Cloning procedures help produce large quantities of DNA for molecular biology studies, such as those of gene structure and expression, for large-scale work, as described in Chapters 10 and 11.
Transformation is the uptake of DNA by bacterial cells.
Bacterial cells can occasionally take up DNA from their medium, and that DNA can survive and replicate in the host cell, especially if it's a plasmid containing an origin of replication recognized by the host.
Not all bacteria are equally efficient at transformation.
To enhance DNA uptake by cells like E. coli, specific treatments (e.g., calcium chloride treatment) are often needed to make them competent.
Transforming competent cells is an inefficient procedure. Only a small proportion of cells actually take up the plasmid DNA during transformation.
Selection of transformed cells typically involves antibiotic resistance genes, where transformed bacteria can grow on selective media containing antibiotics, and non-transformed bacteria cannot.
Recombinants are identified by testing for characteristics coded by inactivated genes.
Some cloning vectors carry a lacZ gene, and the insertion of new DNA into it disables B-galactosidase production, distinguishable by screening with X-gal on agar plates (blue versus white colonies).
Phage DNA can be introduced into bacterial cells via transfection (similar to transformation, but using phage DNA) or in vitro packaging (where phage packaging proteins are used to assemble recombinant phage DNA).
Recombinant phage plaques can be identified by inactivation of a lacZ gene or other strategies resulting in distinctive colors or morphologies.
DNA uptake into yeast, fungal, animal, and plant cells is done via different techniques.

5.1 Transformation—the uptake of DNA by bacterial cells

Most bacterial species can take up DNA.
Occasional DNA uptake and replication occurs, often when plasmids with the host cell's replication origin are involved.
Specific species like Bacillus and Streptococcus can be easily transformed in the lab.
Transformation efficiency can be improved by treating bacteria to make them competent—increasing their ability to take up DNA.

5.1.1 Not all species of bacteria are equally efficient at DNA uptake

Transformation is not a primary method for bacteria acquiring genetic information in nature.
Some bacteria have evolved sophisticated mechanisms for DNA binding and uptake.
Typically, E. coli cells take up limited DNA under normal circumstances.
For enhanced DNA uptake, physical or chemical treatments (to enhance competence) are needed for many bacterial species.

5.1.2 Preparation of competent E. coli cells

A common solution used for E. coli transformation is 50 mM calcium chloride (CaCl2) on ice.
Heating to 42°C (heat shock) causes cells to take up the DNA.
The exact mechanisms of these methods are not fully understood.

5.1.3 Selection for transformed cells

Transformation is typically done with pUC8 (a plasmid vector for creating recombinant DNA) and can result in 1,000-10,000 transformants from 1 nanogram of DNA.
Selection for transformed cells involves screening for expression of genes carried on the plasmid, typically related to antibiotic resistance.
Cells taking up plasmids are capable of growing in media with the antibiotic the plasmid confers resistance to.

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Description

This quiz explores the process of introducing recombinant DNA into living cells, focusing on bacterial transformation. Understand the significance of cloning in producing and purifying DNA for molecular biology studies. Delve into key concepts from previous chapters that contribute to the gene cloning process.