Chapter 21

Which of the following may be used as a vector in a gene-cloning experiment? mRNA Plasmid Virus Both b and c

Both b and c

when a vector is replicated within a host cell the _____ that it carries is also replicated

DNA

Prior to the action of DNA ligase, how many hydrogen bonds are holding these two DNA fragments together?

eight

plasmids

small circular DNA molecules that replicate separately from the bacterial chromosome

The hydrogen bonding between the sticky ends of DNA fragments promotes a temporary interaction between the two fragments. However, this interaction is not stable because it involves only a few hydrogen bonds between complementary bases. How can this interaction be made more permanent?

The answer is that the sugar-phosphate backbones within the DNA strands must be covalently linked together. Experimentally, this linkage is catalyzed by the addition of DNA ligase

Vector

a small segment of DNA that is used as a carrier of another segment of DNA. Vectors are used in DNA cloning experiments a gene is removed from its native site within a chromosome and inserted into a smaller segment of DNA known as a vector—a small DNA molecule that replicates independently of the chromosomal DNA and produces many identical copies of an inserted gene. The purpose of vector DNA is to act as a carrier of the DNA segment to be cloned.

recombinant vector

a vector that contains an inserted fragment of DNA, such as a gene from a chromosome

The restriction enzymes used in gene-cloning experiments ___________, which generates sticky ends that can ___________. cut the DNA, enter bacterial cells cut the DNA, hydrogen bond with complementary sticky ends methylate DNA, enter bacterial cells methylate DNA, hydrogen bond with complementary sticky ends

cut the DNA, hydrogen bond with complementary sticky ends

DNA ligase

enzyme that catalyzes the formation of a phosphodiester covalent bond between adjacent 3'-OH and 5'-phosphate groups in a DNA molecule backbone, after the sticky ends have been hydrogen bonded together

describe the methods used for generating recombinant DNA molecules

isolate and purify pieces of DNA in a test tube and then covalently link DNA fragments from two different sources

The vectors commonly used in gene-cloning experiments were derived originally from two natural sources:

viruses and plasmids

transformation

(1) when a plasmid, vector, or segment of chromosomal DNA is introduced into a bacterial cell (2) when a normal cell is converted into a malignant cell

complementary DNA (cDNA)

DNA that is made from an RNA template by the action of reverse transcriptase

R factors

Genes on plasmids that provide antibiotic resistance

How is reverse transcriptase used in viruses?

This enzyme is encoded in the genome of retroviruses and provides a way for retroviruses to copy their RNA genome into DNA molecules that then integrate into the host cell's chromosomes.

genomic library

a DNA library of recombinant vectors that carry chromosomal DNA fragments

host cell

a cell that is infected with a virus or bacterium, or one that harbors a vector

shuttle vector

a cloning vector that can propagate in two or more different species, such as E. coli and yeast

expression vector

a cloning vector that contains a promoter so that the gene of interest is transcribed into RNA when the vector is introduced into a host cell

DNA library

a collection of many hybrid vectors, each carrying a particular fragment of DNA from a larger source.

A collection of recombinant vectors that carry fragments of chromosomal DNA is called a genomic library. a cDNA library. a Northern blot. either a or b.

a genomic library.

restriction enzymes (restriction endonucleases)

an endonuclease that cleaves DNA The restriction enzymes used in cloning experiments bind to specific base sequences and then cleave the DNA backbone at two defined locations, one in each strand

reverse transcriptase

an enzyme that uses an RNA template to make a complementary strand of DNA

sticky ends

are single-stranded regions of DNA that hydrogen bond to a complementary sequence of DNA from a different source. The ends of two different DNA pieces hydrogen bond to each other because of their complementary sticky ends

competent cells

cells that can take up DNA from an extracellular medium

The function of reverse transcriptase is to copy RNA into DNA. copy DNA into RNA. translate RNA into protein. translate DNA into protein.

copy RNA into DNA.

when are vectors used?

in DNA cloning experiments

plasmids contain a DNA sequence

origin of replication--that is recognized by the replication enzymes of the host cell, which allows the plasmid to be replicated. The sequence of the origin of replication determines whether or not the vector can replicate in a particular type of host cell

what is used to clone large pieces of DNA

other types of vectors, such as cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs), are used to clone large pieces of DNA

Restriction enzymes usually recognize sequences that are

palindromic

most vectors are

plasmids

What was proposed by Werner Arber in the 1960s and discovered by Hamilton Smith and Daniel Nathans in the 1970s?

restriction enzymes are made naturally by many species of bacteria and protect bacterial cells from invasion by foreign DNA, particularly that of bacteriophages

palindromic

sequence in one strand is the same as that in the complementary strand when read in the opposite direction.

origin of replication

site on a chromosome that functions as an initiation site for the assembly of several proteins that begin the process of DNA replication

gene cloning

the process of making many copies of a gene

Which is the proper order of the following steps in a gene-cloning experiment involving vectors? 1 Add DNA ligase. 2 Incubate the chromosomal DNA and the vector DNA with a restriction enzyme. 3 Introduce the DNA into living cells. 4 Mix the chromosomal DNA and vector DNA together. 1, 2, 3, 4 2, 3, 1, 4 2, 4, 1, 3 1, 2, 4, 3

2, 4, 1, 3

selectable marker

A gene that provides a selectable phenotype in a cloning experiment. Many selectable markers are genes that confer antibiotic resistance.

A key step in a cloning experiment is the insertion of chromosomal DNA into a plasmid or viral vector. This requires the cutting and pasting of DNA fragments. What is needed to cut DNA?

restriction enzyme/endonuclease