Click here to
Reproduction
Cellular Life
Tetrad Analysis

Tetrads

How do we know that genes and chromosomes sort themselves out independently of one another as diploid brewer's yeast cells become haploid brewer's yeast cells?

Gregor Mendel showed that all the characters of his pea plants appeared to sort themselves out into their offspring in a way that would suggest that a random process was involved, but he could neither see what was happening in meiosis, nor was he looking at the immediate products of meiosis, his plants needed a further fertilization, and the production of the next generation of diploid plants, before he could see what was happening.

Sudying meiosis in brewer's yeast (Saccharomyces cerevisiae) solves all these problems.

Under the appropriate environmental conditions, a diploid yeast cell will undergo meiosis, separate all their chromosomes into haploid sets once more, and package the results into four, smaller, separate haploid cells which can be clearly seen and micro-manipulated under a microscope.

The tiny cluster of four haploid cells (called "spores") that results from a single round of meiotic division stay together in a structure called an ascus. This makes it possible for the experimenter to identify them as a tetrad of sibling spores. After suitable enzymatic digestion of the ascus wall, each spore can be teased apart using a very fine glass probe. Each of the four haploids cells can then be grown into independent colonies of identical cells that can be studied for the genes they carry.

This is a very powerful technique in genetics which has been used a lot to discover the nature of genes, where they lie on their chromosomes and how many of these genes work in their cells.

tetrad analysis

mutation

A typical genetic experiment with yeast cells starts with mutants. Using a variety of agents, chemicals or environmental factors, the biological information in normal haploid yeast cells is damaged. Most of the damaged cells die, as they can no longer make vital growth factors such as essential amino acids.

With careful technique, however, the experimenter can save some of these mutants by providing them with the missing ingredients they cannot now make for themselves.

selection

Normal yeast cells grow on a very minimal media (supply of food) which contains little else than sugar. Yeast use the sugar as a source of energy and make almost everything else they need, including the amino acid tryptophan.

Mutants that cannot make tryptophan for themselves must be supplied with this essential amino acid in their diet, or they will not grow.

It is quite easy to spot such mutants. Colonies of normal yeast grow on both minimal media (just sugar) and on media supplemented with additional tryptophan. Mutants, on the other hand, will only grow on media supplemented with the extra tryptophan. They will not grow on the minimal media.

mating haploids

Haploid yeast cells have two different 'mating types', which are roughly equivalent to different sexes.

Haploid yeast of the mating type 'a' will fuse with and join up with haploid yeast of the mating type 'alpha, to form a larger and diploid yeast cell which does not normally mate further.

Diploid yeast cells can usually be selected out of the mating mixture and grown in their own media for further analysis.

meiosis and tetrad formation

Diploid yeast cells can be made to undergo meiosis by growing them on a special, starvation, media. The diploids divide up their chromosomes and DNA into 4 separate packages, form a nucleus around each and then enclose each nucleus in a resistant wall or coat.

However, the four products of each meiosis more or less stay together, trapped in the wall of the old, parental cell. An experimenter can therefore treat a dilute solution of these post-meiotic cells (with their 4 separate haploids), with an enzyme that digests the old wall.

If this is done very carefully, it is possible to see all 4 haploids from one meiosis, still all together, under a microscope. All the experimenter has to do is gently separate out the 4 haploids onto their own, clean, sterile media and allow them to grow separately.

When each haploid has grown into a tiny colony of cells, the genetic composition of each can be determined by observing what kind on media it will grow on. Those cells carrying mutant genes will still not grow on minimal media, but those cells with all normal genes will grow on minimal media with no difficulty.

keeping good records

It is important that the experimenter keep good records of all the stages of the tetrad analysis experiment listed above.

The nature of the mutation must be recorded (i.e. what function is missing from the mutant yeast). Each mutant is usually given a number and some sort of name that indicates what is missing.

For example, a yeast that cannot grow without additional tryptophan is termed:

try- (pronounced - "trip minus")

Many different mutants of this yeast strain may be "trip minus", so each mutant is given a different number. The experimenter usually makes up a numbering system, but one possible example could be:

1123c - try-

1125a - try-

1226b - try-

2043d - try-

The numbers themselves might refer to the order in which these mutants were selected, the date they were found or the number of the petri dish on which they were discovered. There are many different systems, but so long as each mutant has its own unique identifying code, they all work.

After mating different permutations and combinations of each haploid mutant, each different diploid produced must be recorded. Usually these are cells which have no deficiencies in their growth requirements, so they are easy to separate from the mating mixture. Once again, each diploid is given a unique identifying number or name.

After the diploid has been grown and induced to form haploids, good records must be kept of every set of cells produced from a single diploid. The growth behavior of each individual cell from each tetrad must be recorded for later analysis.


BIOdotEDU
© 2002, Professor John Blamire