Microbiology and Immunology

WORLD OF MICROBIOLOGY AND IMMUNOLOGY Signal hypothesis

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food. Both are routes of transfer of fecal material to the food.

The amount of fecal material need not be great, as studies have

proven that only 10 living Shigellaare required to establish an

infection in humans.

The infection tends to be fairly short in duration and

clears without any therapeutic intervention. In some people,

however, the primary infection can be the prelude to very

damaging infections of the kidney and the joins. The latter

infection, which is caused by Shigella flexneri, is known as

Reiter’s syndrome. This can persist for years. During this time,

infections by other strains of Shigellaare possible.

Shigellosis results from the attachment of the bacteria to

epithelial cells that line the intestinal tract, and the entry of the

bacteria into the cells. Within the host cells, the bacteria divide

and can then spread laterally to infect other host cells. The

interior location of the bacteria protects them from any host

immune response or from antibiotics. Additionally, some

strains of Shigellaproduce the toxins that can damage the

epithelial cells.

The establishment of an infection is easier in people

whose immune systems are compromised. For example,

shigellosis is a significant problem in those afflicted with

acquired immunodeficiencysyndrome.

Treatment for Shigellainfections is not always clini-

cally prudent. Many infections, while very inconvenient and

painful, pass relatively quickly. Management of the symp-

toms, particularly ensuring proper hydration, is preferred in

immunocompetent people, as opposed to antibiotic therapy.

The reason is that the bacteria can rather readily acquire resist-

ance to antibiotics, which can make eradication of the bacteria

even harder. Also, the antibiotic resistant bacteria can be

excreted in the feces of the infected individual, and may then

spread the resistant strain to other people.

Prevention of the spread of infection involves proper

hygiene and thorough cooking of foods.

See alsoEnterobacteriaceae; Enterotoxin and exotoxin; Food

safety

SShotgun cloningHOTGUN CLONING

The shotgun method (also known as shotgun cloning) is a

method in cloning genomic DNA. It involves taking the DNA

to be cloned and cutting it either using a restriction enzyme or

randomly using a physical method to smash the DNA into

small pieces. These fragments are then taken together and

cloned into a vector. The original DNA can be either genomic

DNA (whole genome shotgun cloning) or a clone such as a

YAC(yeast artificial chromosome) that contains a large piece

of genomic DNA needing to be split into fragments.

If the DNA needs to be in a certain cloning vector, but

the vector can only carry small amounts of DNA, then the

shotgun method can be used. More commonly, the method is

used to generate small fragments of DNA for sequencing. A

DNA sequence can be generated at about 600 bases at a time,

so if a DNA fragment of about 1100kb is cloned, then it can be

sequenced in two steps, with 600 bases from each end, and a

hundred base overlap. The sequencing can always be primed

with a known sequence from the vector and so any prior

knowledge of the sequence that has been cloned is not neces-

sary. This approach of shotgun cloning followed by DNA

sequencing from both ends of the vector is called shotgun

sequencing.

Shotgun sequencing was initially used to sequence

small genomes such as that of the cauliflower mosaic virus

(CMV), which is 8kb long. More recently, it has been applied

to more complex genomes. Usually this involves creating a

physical map and a contig (line of overlapping clones) of

clones containing a large amount of DNA in a vector such as

a YAC, which are then shotgun cloned into smaller vectors and

sequenced. However, a whole genome shotgun approach has

been used to sequence the mouse, fly and human genomes by

the private company Celera. This involves shotgun cloning the

whole genome and sequencing the clones without creating a

physical map. It is faster and cheaper than creating a physical

genemap and sequencing clones one by one, but the reliabil-

ity of reassembling all the sequences of the small fragments

into one genomic sequence has been doubted. For example, a

part of the fly genome was sequenced by the one-by-one

approach and the whole genome shotgun method. The two

sequences were compared, and showed differences. 60% of

the genes were identical, 31% showed minor differences and

9% showed major differences. The whole genome shotgun

method generated the sequence much more quickly, but the

one-by-one approach is probably more accurate because the

genes were studied in more detail.

See alsoCloning, application of cloning to biological prob-

lems; Yeast artificial chromosome (YAC); Yeast genetics

SSignal hypothesisIGNAL HYPOTHESIS

The signal hypothesis was proposed to explain how proteins

that were destined for export from bacteriaor for targeting to

certain regions within eucaryotic microorganisms(e.g., yeast)

achieved their target. The hypothesis was proposed in the

1970s by Günter Blobel, who was then as now a molecular

biologist at the Rockefeller University in New York. Blobel’s

work received the 1999 Nobel Prize in medicine or physiology.

The signal hypothesis proposes that proteins destined

for secretion, which involves the movement of the protein

across a biological membrane, are originally manufactured

with an initial sequence of amino acids that may or may not

present in the mature protein.

Work by Blobel and others over two decades estab-

lished the validity of the proposal. The so-called signal

sequence is now known to be only some 20 amino acids in

length. The arrangement of amino acids in the signal

sequence is not random. Rather, the beginning of the

sequence, along with a few amino acid residues in the center

of the sequence, is comprised of amino acids that are

hydrophilic (“water-loving”). Sandwiched between these

regions is a central portion that is made up of amino acids that

are hydrophobic(“water-hating”).

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