Hogg S. Essential microbiology

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328 MICROORGANISMS IN GENETIC ENGINEERING

Multiple

cloning site

Terminator

Selectable

marker

ori

Ribosomal

binding site

Operator

Promoter

Figure 12.11 A cassette vector. In order for a foreign gene to be transcribed and translated

successfully, it must be surrounded by the appropriate expression sequences. In a cassette

vector, these sequences ﬂank the RE recognition sites at which the foreign DNA is inserted

Another obstacle to the cloning of such proteins concerns a fundamental difference in

the way that procaryotic and eucaryotic systems convert the message encoded in DNA

to messenger RNA (see Chapter 11). Procaryotes lack the means to remove introns, so

Complementary DNA

(cDNA) is DNA pro-

duced using mRNA as a

template, using the en-

zyme reverse transcrip-

tase. It represents only

the coding sequences

of the parent DNA mole-

cule.

if a human gene, for example, is expressed, the whole

of the primary transcript will be translated, instead of

just the coding sequences, leading to a non-functional

protein. This problem can be circumvented by cloning

not the entire gene, but its cDNA, that is, just those DNA

sequences that are transcribed into mRNA and subse-

quently translated into amino acid sequences. This can

be done by isolating mRNA, then using reverse transcrip-

tase to make a DNA copy. In the case of proteins such as

insulin, the very small size enabled artiﬁcial genes to be

synthesised, based on their known amino acid sequences.

Eucaryotic cloning vectors

A number of problems are associated with the expression of complex eucaryotic genes

in bacterial cloning systems, some of which have just been outlined. In order to avoid

these, such genes may be expressed in eucaryotic cloning systems, the model system

for which is the yeast Saccharomyces cerevisiae. This holds many attractions for the

experimenter:

it is safe and easy to handle in the laboratory

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EUCARYOTIC CLONING VECTORS 329

Table 12.1 Characteristics of yeast cloning vectors

Plasmid type Abbreviation Features

Yeast episomal plasmid YEp Contain 2 µm circle (naturally occurring yeast

plasmid) origin of replication and whole of

pBR322 sequence. Can integrate into yeast

chromosomal DNA or replicate independently.

High copy number

Yeast integrative plasmid YIp Bacterial plasmid containing yeast selectable

marker. Stably integrates into yeast

chromosome. Unable to exist independently in

yeast. Low transformation rate and single copy

number

Yeast replicative plasmid YRp Carries yeast chromosomal origin of replication

(ARS); high copy number; unstable

Yeast centromere plasmid YCp Contain centromere sequence – ensures stability,

but low (single) copy number

it grows at higher density than E. coli

it grows much faster than other eucaryotic cells in culture (e.g. cultured mam-

malian cells)

Several types of vector have been developed for use in yeasts. These are summarised in

Table 12.1. The choice of which type to use will depend on the relative importance to a

particular application of factors such as yield and long-term stability. Most yeast vectors

are shuttle vectors, that is, vectors that can be replicated in more than one type of host

cell (in this case, yeast and E. coli). Initial cloning is more easily done in E. coli, then

the recombinant vector is transferred to yeast cells, which can carry out functions such

as protein folding and glycosylation that are not possible in bacteria. Shuttle vectors

must contain selectable markers and an origin of replication (or means of integration)

for both cell types.

YACs, BACs and PACs

The centromere is the

central region of the

chromosome that en-

sures correct distribution

of chromosomes be-

tween daughter cells

during cell division, whilst

telomeres are important

in preserving the stability

of the tips of each chro-

mosome arm.

We saw earlier in this chapter how the use of cosmid

vectors extended the size of cloneable insert to around

45kb. Many eucaryotic genes, however, are bigger than

this, and so cannot be cloned in a single sequence us-

ing such a system. In addition, the ability to clone large

DNA fragments is essential for the physical mapping

of complex eucaryotic genomes such as that of humans

(3 × 10

bp). Yeast artiﬁcial chromosomes (YACs) have

been developed to accommodate insert fragments of up

to 1Mb (1000kb). They contain key sequences from a

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330 MICROORGANISMS IN GENETIC ENGINEERING

EcoRI

Genomic DNA

Partial digest

with EcoRI

EcoRI

EcoRI EcoRI

Genomic

DNA

Mix and ligate

EcoRI

AMP

pYAC4

SUP4

ARS2

URA3

CEN4

TRP1

URA3

TRP1

ori

CEN4

TRP1

CEN4

TEL

BamHI BamHI

BamHI

Cut with EcoRI

and BamHI

TEL

Figure 12.12 Yeast artiﬁcial chromosomes (YACs) are used to clone large fragments of

DNA. Cleavage with the REs BamHI and SnaBI leads to the formation of two arms, between

which fragments of foreign DNA of several hundred kb may be ligated. The ﬁnal construct

contains a centromere, telomere sequences and an origin of replication, and is able to act

like a natural chromosome. From Reece, RJ: Analysis of Genes and Genomes, John Wiley &

Sons, 2003 Reproduced by permission of the publishers

yeast chromosome that ensure its stable replication; these include an origin of replica-

tion (the autonomous replication sequence, ARS) the CEN sequence from around the

centromere, and the telomeres at the end of the chromosome. When placed in S. cere-

visiae cells, the presence of these sequences allows the YAC to replicate along with the

natural chromosomes.

The fragment to be cloned is inserted between the two arms of the YAC (Figure 12.12).

Each arm carries a selectable marker; it is important to have two, to ensure that each

construct comprises both a right and a left arm, and not two of the same. Insertional

inactivation of a third selectable marker (situated around the point of insertion) allows

the detection of recombinants.

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EUCARYOTIC CLONING VECTORS 331

Bacterial artiﬁcial chromosomes (BACs) allow the cloning of fragments as large as

300kb in length into E. coli host cells, although 100–150kb is more routinely used. They

are based on the naturally occurring F plasmid of E. coli; recall from our description of

bacterial conjugation in Chapter 11 that the F plasmid can pick up considerable lengths

of chromosomal DNA, when it becomes known as F’. BACs have the advantage over

YACs that they are easier to manipulate, and inherently more stable.

Phage P1-derived artiﬁcial chromosomes (PACs) are another relatively recently de-

veloped class of vector for use in E. coli, with a comparable capacity to that of BACs.

Viruses as vectors in eucaryotic systems

Several proteins of clinical or commercial interest are too complex to be expressed using

microbial host cells, even eucaryotic ones, and are only properly produced in mammalian

systems. Vectors based on animal viruses such as SV40, adenoviruses and vaccinia virus

have been successfully developed for use in these. Vaccinia has been particularly valuable

in the development of recombinant vaccines.

Viruses of humans such as adenoviruses and retroviruses have also been tested as

vectors in the exciting new technique of gene therapy, which attempts to ameliorate the

effects of genetic disorders by introducing the ‘correct’ form of the defective gene into

the patient’s cells. Here it is important to ensure stable integration of the inserted DNA

into the host chromosome.

A large virus that infects insects, the baculovirus, has been found to be a highly

efﬁcient vector for the large-scale expression of eucaryotic proteins in cultured in-

sect cells. The rate of expression is much higher than in cultured mammalian cells,

and the necessary protein folding and post-translational modiﬁcations are correctly

executed.

Cloning vectors for higher plants

The most important single tool for the genetic engineering of plants is the Ti plasmid.

This is found naturally in the soil bacterium Agrobacterium tumefaciens, which infects

plants at wound sites, and leads to a condition called crown gall disease. The impor-

tant feature of this plasmid is that part of it, called the T-DNA, can integrate into the

host plant’s chromosomes, and be expressed along with host genes (Figure 12.13). Ge-

neticists were quick to spot the potential of the Ti plasmid, replacing tumour-forming

genes with foreign genes, and having them expressed in plant tissues. The recombinant

A. tumefaciens is used to infect protoplasts, which can be regenerated into a whole plant,

every cell of which will contain the integrated foreign gene. The Ti plasmid system has

been used in the successful transfer of genes for insect- and herbicide-resistance into

economically signiﬁcant crop plants.

Plant viruses have very limited usefulness as vectors. Only the caulimoviruses and the

geminiviruses have DNA rather than RNA as their genomic material, and a variety of

problems, including instability of inserts and narrow host range, have been encountered

with the use of these.

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332 MICROORGANISMS IN GENETIC ENGINEERING

Auxin

synthesis

Opine

synthesis

Opine

utilisation

ori

Virulence genes

Cytokinin

synthesis

(a)

T DNA

plasmid

Wound

Invasion by

Agrobacterium

Formation of

crown gall

Opine

production

(

)

Figure 12.13 Agrobacterium tumefaciens. (a) The Ti plasmid. The T-DNA contains genes

for tumour production and the synthesis of opines, unusual amino acid derivatives that

serve as nutrients for A. tumefaciens. (b) Crown gall formation by A. tumefaciens. The

T-DNA integrates into the DNA of the host, where its genes are expressed. This ability has

been exploited in order to transfer foreign genes into plant cells. From Reece, RJ: Analysis of

Genes and Genomes, John Wiley & Sons, 2003. Reproduced by permission of the publishers

Genetically engineered insect resistance using Bacillus thuringiensis

Destruction of crops by insect pests is a huge problem throughout the world, and the

use of chemical insecticides is only partially successful in countering it. The drawbacks

to the use of such chemicals are manifold:

they are often non-speciﬁc, so beneﬁcial insects as well as harmful ones are

killed

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POLYMERASE CHAIN REACTION (PCR) 333

they are often non-biodegradable, so they can have lasting environmental ef-

fects

aerial spraying only reaches upper leaf surfaces

resistance develops with continued use.

A form of natural insecticide does exist; it is a crystalline protein produced during

sporulation by Bacillus thuringiensis. This is highly toxic to insects when converted

to its active form by the enzymes of the gut. This δ-endotoxin is relatively selective;

different strains of B. thuringiensis produce different forms of the toxin, which are

effective against the larvae of different insects. Surprising as it may seem, the use of the

δ-endotoxin as an insecticide was patented a hundred years ago; however, the success

of it has been limited due to a variety of practical considerations.

The development of genetic engineering techniques has meant that attention has

turned in more recent times to introducing the genes for the δ-endotoxin into the crops

themselves, so that they synthesise their own insecticide. This has been achieved with

some success, but the problem of the insects building up resistance remains.

As a spin-off from the Human Genome Project, the genetic make-up of many microor-

ganisms has been elucidated. One of these is Photorhabdus luminescens, which encodes

a toxin lethal to the two species of mosquito responsible for the spread of malaria, and

it is hoped that determining the sequence of the gene will lead to applications in insect

control.

Polymerase chain reaction (PCR)

First described in the mid-1980s by Kary Mullis, PCR is probably the most signiﬁcant

development in molecular biology since the advent of gene cloning. PCR allows us to

amplify a speciﬁc section of a genome (for example a particular gene) millions of times

PCR selectively repli-

cates a specific DNA se-

quence by means of in

vitro enzymatic amplifi-

cation.

from a tiny amount of starting material (theoretically

a single molecule!). The impact of this powerful and

highly speciﬁc process has been felt in all areas of biology

and beyond. Medicine, forensic science and evolutionary

studies are but three areas where PCR has opened up

new possibilities over the last 15 years or so. To appre-

ciate how PCR works, you will need to understand the

role of the enzyme DNA polymerase in DNA replication,

as outlined in the previous chapter. This is the enzyme,

you’ll recall, that when provided with single-stranded

DNA and a short primer, can direct the synthesis of a

complementary second strand. Figure 12.14 illustrates

the three steps in the PCR process:

A primer is a short nu-

cleotide sequence that

can be extended by

DNA polymerase. In

PCR, synthetic primers

are designed to flank ei-

ther side of the target se-

quence.

Denaturation: by heating to 95

◦

C, the DNA is sepa-

rated into single strands, providing a template for the

DNA polymerase.

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334 MICROORGANISMS IN GENETIC ENGINEERING

3’5’

5’3’

5’

3’

Primers

3’

5’

3’

1. Denature at 95

2. Anneal primers at 50-60

3’

5’

3’

5’

3’

3. Extend primers at 72

hermostable DNA

polymerase

Figure 12.14 The polymerase chain reaction (PCR). Millions of copies of a speciﬁc se-

quence of DNA can be made by PCR. Samples are subjected to repeated cycles of denatu-

ration, annealing of primers and extension by a thermostable DNA polymerase (for details

see the text). At the end of the ﬁrst cycle (shown in the diagram), each of the four strands

can serve as a template for replication in cycle two. After 30 cycles, over a billion copies of

the target sequence will have been made

Primer annealing: at a lower temperature (typically 50–60

◦

C, the exact value de-

pends on the primer sequence), short single-stranded primers are allowed to attach.

Primers are synthetic oligonucleotides with a sequence complementary to the regions

ﬂanking the region we wish to amplify. One primer anneals to each strand, at either

side of the target sequence.

Polymerase extension: at around 72

◦

C, the DNA polymerase extends the primer by

adding complementary nucleotides and so forms a second strand.

The net result of this process is that instead of one double-stranded molecule, we now

have two. We now come to the key concept of PCR; if we raise the temperature to 95

◦

again to start another cycle, we will have not two, but four single-stranded templates to

work on, each of which can be converted to the double-stranded form as before. After

20 such cycles we should have, in theory, over a million copies of our original molecule!

Typical PCR protocols run for 30–35 cycles. All this can be achieved in just a couple

of hours; the temperature cycling is carried out by a programmable microprocessor-

controlled machine called a thermal cycler.

PCR has widespread applications in microbiology, as in other ﬁelds of biology, but

in addition, microorganisms play a crucial role in the process itself. If you have read the

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TEST YOURSELF 335

above description of PCR carefully, something should have struck you as being not quite

right: how can the DNA polymerase (a protein) tolerate being repeatedly heated to over

◦

C, and what sort of an enzyme can work effectively at 72

◦

C? The answer is that

the DNA polymerase used in PCR comes from thermophilic bacteria such as Thermus

aquaticus, a species found naturally in hot springs. The optimum temperature for Taq

polymerase is 72

◦

C, and it can tolerate being raised to values considerably higher than

this for short periods. The availability of this enzyme meant that it was not necessary

to add fresh enzyme after each cycle, and the whole process could be automated, a key

factor in its subsequent phenomenal success.

Test yourself

1 are enzymes that cut DNA at speciﬁc se-

quences in the

backbone.

2 Joining together DNA fragments from different sources produces a

molecule.

3 DNA fragments with

tend to join together because of

their complementary single-stranded overhangs.

4 In order to gain access to a host cell and be replicated, a fragment of DNA

must be taken up by a

molecule.

enable us to detect which host cells have been trans-

formed. A commonly used example is resistance to

. By insert-

ing foreign DNA at this point, recombinants can be detected by

6 Another name for a polylinker is a

7 A short sequence of labelled single-stranded DNA used to locate a speciﬁc

target sequence is known as a

8 The removable central section of the phage Lambda genome is known as a

fragment.

9 Bacteriophage

is a useful vector if DNA is required in a

form.

10 Cloning vectors combining features of plasmids and phage Lambda are called

vectors incorporate foreign DNA at a site that is surrounded by

essential signal sequences.

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336 MICROORGANISMS IN GENETIC ENGINEERING

12 E. coli is not a suitable host for the expression of complex eucaryotic proteins

because it lacks the molecular machinery to carry out

modiﬁcations

such as

DNA contains only those sequences that are involved in coding

for a polypeptide product.

14 Yeast plasmids are based on a naturally occurring sequence called the

vectors are able to replicate in both and cells.

16 A virus found to be useful in the expression of eucaryotic genes is the

. It is grown in cultured cells.

17 The soil bacterium A. tumefaciens contains a plasmid called

, which

causes crown gall disease. Part of it, the

, integrates into host

chromosomes.

18 Genes from Bacillus thuringiensis coding for

have been introduced

into plant cells, making them

resistant.

19 The polymerase chain reaction (PCR) comprises repeated cycles of the follow-

ing three steps:

, of primers and by

DNA polymerase.

20 PCR results in an

increase in the number of copies of the target

sequence.

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Part V

Control of Microorganisms

337