Microbiology and Immunology

Fungal genetics WORLD OF MICROBIOLOGY AND IMMUNOLOGY

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contaminationfrom airborne bacteriaor viruses. The latter is of

particular relevance in some viral research, where the tissue sur-

faces used to grow the virus are prone to contamination.

The design of fume hoods differs, depending on the

intended purpose (general purpose, chemical, radioisotope,

biological). But all fume hoods share the feature of an

inward flow of air. In biological fume hoods the flow of ster-

ile air is typically from the back of the cabinet toward the

laboratory worker, and from the top of the fume hood down-

ward across the opening at the front of the hood. This pattern

of airflow ensures that any microorganismsresiding on the

laboratory worker are not carried into the work surface, and

that no air from inside the cabinet escapes to the outside of

the cabinet. Any air that is exhausted back into the laboratory

first passes through filters that are designed to remove bio-

logical and viral contaminants. The most popular type of bio-

logical filter is the high-energy particulate air (or HEPA)

filter.

Biological fume hoods can have a moveable, protective

glass partition at the front. Most hoods also have a gas source

inside, so that sterile work, such as the flaming of inoculation

loops, can be done. The fume hood should be positioned in an

area of the laboratory where there is less traffic back and forth,

which lessens the turbulence of air outside the fume hood.

The filtering system of biological fume hoods restricts

its use to biological work. Work involving noxious chemicals

and vapors needs to be conducted in another, specially

designed chemical fume hood.

The construction of fume hoods is conducted according

to strict protocols of safety and performance monitoring. In

normal laboratory use, the continued performance of a fume

hood is regularly monitored and test results recorded. Often

such checks are a mandatory requirement of the ongoing cer-

tification of an analysis laboratory. Accordingly, laboratories

must properly maintain and use fume hoods to continue to

meet operating rules and regulations.

See alsoBioterrorism, protective measures; Containment and

release prevention protocol

FFungal geneticsUNGAL GENETICS

Fungipossess strikingly different morphologies. They include

large, fleshy, and often colorful mushrooms or toadstools, fil-

amentous organisms only just visible to the naked eye, and

single-celled organisms such as yeasts. Molds are important

agents of decay. They also produce a large number of indus-

trially important compounds like antibiotics(penicillin, grise-

ofulvin, etc.), organic acids (citric acid, gluconic acid, etc.),

enzymes(alpha-amylases, lipase, etc.), traditional foods (soft-

ening and flavoring of cheese, shoyu soy sauce, etc.), and a

number of other miscellaneous products (gibberellins, ergot

alkaloids, steroid bioconversions). As late as 1974 the only

widely applicable techniques for strain improvement were

mutation, screening, and selection. While these techniques

proved dramatically successful in improving penicillin pro-

duction, they deflected attempts to employ a more sophisti-

cated approach to genetic manipulation. The study of fungal

genetics has recently changed beyond all recognition.

The natural genetic variation present in fungal species

has been characterized using molecular methods such as elec-

trophoretic karyotyping, restriction fragment length polymor-

phism, DNAfinger printing, and DNA sequence comparisons.

The causes for the variation include chromosomal polymor-

phism, changes in repetitive DNA, transposons, virus-like

elements, and mitochondrial plasmids.

Genetic recombinationoccurs naturally in many fungi.

Many industrially important fungi such as Aspergilli and

Penicillialack sexuality, so in these species parasexual systems

(cycles) provide the basis for genetic study and breeding pro-

grams. The parasexual cycle is a series of events that can be

induced when two genetically different strains are grown

together in the laboratory. A heterokaryon, which is mycelium

with two different nuclei derived from two different haploid

strains, is produced by the fusion of hyphae. Increased peni-

cillin titer in the haploid progeny of parasexual crosses has been

achieved in Penicillium chrysogenum.A more direct approach

has been developed using protoplasts. These are isolated from

vegetative cells of fungi or yeasts by removing the cell wall by

digestion using a cell wall degrading enzyme. Protoplasts from

the two strains can be fused by treatment with polyethylene gly-

col. Protoplast fusion in fungi initiates the parasexual cycle,

resulting in the formation of diploidy and mitotic recombination

and segregation. A selection procedure to screen such fusants is

done using genetic markers. A good example of applying this

technique is the fusion of a fast growing but poor glucoamylase

producer with a slow growing but excellent producer of glu-

coamylase. The desired result will be a strain that is both fast

growing and an excellent producer of enzyme.

The realization that transformationof genetic material

into fungi can occur came with the discovery that yeasts like

Saccharomyces cerevisiae and filamentous fungi like

Podospora anserinecontain plasmids. Currently transforma-

tion technology is largely based on the use of Neurospora

crassaand Aspergillus nidulans,though methods for use in

filamentous organisms are being developed. The protocols

used in transformation of filamentous fungi involve cloning

the desired geneinto the plasmid from E. colior a plasmid

constructed from genetic material from E. coli and

Saccharomyces cerevisiae. Protoplasts from the recipient

strains are then formed and mixed with the plasmid. After

incubating for a short time to allow for the uptake of the plas-

mid DNA, the protoplasts are allowed to regenerate and the

cells are screened for the presence of the specific marker.

The application of recombinant DNA to yeasts and fila-

mentous fungi has opened up new possibilities in relation to

the construction of highly productive strains. The filamentous

fungi are now established as potent host organisms for the pro-

duction of heterologous proteins. This is particularly useful as

expression of specific proteins can reach relatively high levels.

Using Aspergillusas a host for reproduction has led to the pro-

duction of many recombinant products like human therapeutic

proteins, including growth factors, cytokines, and hormones.

While expression can be good in E. coli,lack of posttransla-

tional modifications has limited their usage. The use of

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