FINE STRUCTURE OF GENES
MODERN CONCEPT OF GENE
The fine structure of a gene is the linkage map of its various mutant alleles. In this map, the sites of mutational changes in different alleles of the same gene are determined on the basis of the frequency of recombination among these alleles. Thus the fine structure map of a gene is essentially comparable to the linkage map of a chromosome [except for the number of genes involved (one in the case of gene fine structures and several in the case of chromosome maps)]. Understandably, the values of crossing over observed in the studies on fine structure of a gene are very low (e.g., 0.01 or even 0.0001%). Therefore extremely large progeny populations have to be effectively screened for efficient identification of the cross over products.
For this reason, fine genetic analysis is more convenient in prokaryotes than in eukaryotes.
THE FINE STRUCTURE OF A GENE IN A PROKARYOTE
The most extensive fine structure map of a locus constructed to date is that of the R Il locus of T4 phage of E.coli due to Seymour Benzer. T4 is similar to T2 in morphology, is an obligate parasite like all viruses, and contains a chromosome of about 200,000 bp (base pairs) long, which is packed within its head. When a T4 phage particle infects a cell of £. coli the bacterial cell lyses in about 20 - 25 minutes liberating 200 -300 progeny phage particles.
When E. coli cells are plated (in an agar medium in petriplates) in sufficiently large numbers they produce a uniform confluent growth or ‘lawn’. If individual T4 particles are placed on the surface of an agar medium seeded with a lawn of E. coli cells, each phage particle would initiate a chain of infection-lysis so that all the E. coli cells in the immediate vicinity of the phage particles will be lysed. This leads to the development of clear zones, called plaques, the lawn of bacterial cells. The plaques produced by the wild type T. particles are relatively small with fuzzy or turbid margins called halos. The halos are produced due to a phenomenon called lysis inhibition,which is a delay in the lysis of T4-infected E. coli cells as a consequence of its subsequent infection by another T4 particle.
The R" LOCUS
Several mutants of T, do not exhibit lysis inhibition, and produce relatively large plaques with clear margins, these mutants are called rapid lysis mutants and are denoted by r. Most of the r mutants map in one of the three distinct loci called r', r! and r@. Mutants in the r! locus are easily recognized due to their inability to multiply in E. coli strain Ki2 (A), which has the chromosome of phage A integrated, in its chromosome. However, r! mutants grow rapidly in other strains of E. colie.g., strain B and K12 (lacking the i. chromosome). Thus r! mutants are conditional lethal as they are unable to grow in Ki2 (A) ; this property was exploited by Benzer for a genetic analysis of r! locus.
COMPLEMENTATION TEST
Benzer isolated over 3000 independent mutants of the rlI locus and subject them to complementation test. For this purpose, cells of E. coli strain K12 (A) were infected with a mixture of the two rll mutants to be tested. Free phage particles not involved in infection were removed from the suspension of bacterial cells through centrifugation. Many bacterial cells will be infected by particles of both the mutants. If there is complementation between the two rlII mutants, the phages will multiply in such host cells and lyse them releasing progeny phage particles. However, if there was no complementation, they will fail to multiply and no progeny phage particles will be released.
A suspension of E. coli Ki2 (A) cells infected in this manner is kept for about 90-120 minutes to permit phage multiplication and bacterial-cell lysis samples from the whole suspension or its supernatant are now placed on E. coli strain b lawn to detect the presence of phage particles. “If plaques develop on £. coli B lawn, it reveals complementation between the two r! mutants used for co-infection; while the absence of plaques signifies a lack of complementation”.
Benzer placed two r! mutants which failed to complement each other in two arbitrary groups designated as A and B. Each of the rl mutants showed complementation either with the mutant belonging to group A or with that belonging to group B, but never with both. Thus all r! mutants could be classified into two clear cut groups (group A and B); mutants of one group showed complementation with the mutants of the other group, but mutants within a group failed to complement each other. Thus the r!! locus was divided into two cistrons, cistron A (r! A, represented by the r! mutant of group A) and cistron B (r! B evidenced by the r! mutants of group B) on the basis of complementation test.
THE ULTIMATE FINE STRUCTURE OF GENE
“The complementation test resolves a locus into distinct cistrons, while the recombination test maps different mutants within each of the cistrons”. The recombination mapping, however, relates to only those regions of a gene that are involved in coding of amino acids of the concerned polypeptide (called exons for expressed sequences). But most eukaryotic genes have one or more intervening non- coding sequences (called introns) within their genes. Such features of the genetic fine structure are revealed only by partial or total determination of the nucleotide sequence of a gene, which constitutes the ultimate fine structure of a gene. Introns and exons are collectively known as split genes since their coding sequences are split into several parts due to the introns.
JUMPING GENES
Most genes reside at a specific locus or position on the chromosome. Some genes or closely linked sets of genes can mediate their own movement from one location to another location in a chromosome. These elements are called as "jumping genes," "mobile elements." "cassettes," "insertion sequences," and "transposons." The formal name for this family of mobile genes is transposable elements, and their movement is called transposition.
NATURE OF TRANSPOSONS
Transposable elements were first discovered in maize and later in phages, bacteria, fungi, insects, viruses and human beings. Barbara McClintock was awarded the Nobel Prize in 1983 for the discovery of transposons in maize. They make up at least 50% of human DNA. Most transposable elements are able to insert at different locations and cause mutations either by inserting into another gene or by promoting DNA rearrangements such as deletions, duplications and inversions.
The transposable elements of bacteria is grouped into
1. Simple transposons (Insertion sequences, or IS) carry only the genetic information necessary for their transposition and
2. Complex transposons contain additional genetic material unrelated to transposition.
STRUCTURE OF A TRANSPOSON
Most transposons have short direct repeats of 3 to 12 base pairs on both sides. They are not a part of a transposable element and do not travel with it but are generated in the process of transposition, at the point of insertion. Also, they have the presence of identical, inverted terminal repeat sequences of 8-38 base pairs. The presence of flanking direct repeats indicates that staggered cuts are made in the target DNA when a transposable element inserts itself. The staggered cuts leave short, single-stranded pieces of DNA on either side of the transposable element. Replication of the single-stranded DNA then creates the flanking direct repeats.
At the ends of transposable elements are terminal inverted repeats, which are sequences from 9 to 40 bp in length that are inverted complements of one another. Each type of transposon has its own unique inverted repeat. The sequence into which a transposable element inserts is called the target sequence.
MECHANISM OF TRANSPOSITION
Transposition refers to the movement of a transposable element from one location to another. Two models_ of transposition in prokaryotes have been proposed, on the basis of the fate of the donor site.
1. In non-replicative transposition, the transposon might be excised from the donor site, leaving no copy of itself at the donor site. It is also called as conservative model.
2. In replicative transposition, the transposon might be replicated, allowing one copy to transpose to another site and leaving an identical copy at the donor site. The replicative mode could produce multiple copies at various sites in the genome. In bacteria, the number of copies of a transposon appears to be regulated, seldom exceeding 20 copies per genome. In eukaryotes, however, the copy number can be very high.
CHROMOSOME WALKING
In chromosome walking, a gene is first mapped in relation to a previously cloned gene. A probe made from one end of the cloned gene is used to find an _ overlapping clone, which is then used to find another overlapping clone. In this way, it is possible to walk down the chromosome to the gene of interest.
The basis of chromosome walking is the fact that a genomic library consists of a set of overlapping DNA fragments. We start with a cloned gene or DNA sequence that is close to the new gene of interest so that the “walk” will be as short as possible. One end of the clone of a neighboring gene (clone A in Figure) is used to make a complementary probe. This probe is used to screen the genomic library to find a second clone (clone B) that overlaps with the first and extends in the direction of the gene of interest. This second clone is isolated and purified and a probe is prepared from its end. The second probe is used to screen the library for a third clone (clone C) that overlaps with the second. In this way, one can walk systematically toward the gene of interest, one clone at a time. A number of important human genes and genes of other organisms have been found in this way.