Discovery of Tranposable Elements in Corn (Zea mays)

• Self pollination of plants known to be true-breeding for pigmented corn kernels yielded the following unexpected ratio of corn kernel offspring:
  12 pigmented : 3 dotted : 1 unpigmented

  Rhodes first hypothesized that two events had occurred at unlinked loci:

  1. pigment gene A₁ had mutated to a colorless mutant, a₁

  2. At another locus, a dominant allele for dotting (Dt) had appeared.

A second hypothesis was proposed:

The original parent plant must have been A₁ A₁ dt dt, and the plant resulting from the mutations was A₁ a₁ Dt dt.

This still didn't explain the dotted kernels. Could the dots have resulted from somatic mutations? Maybe...but there would have had to be a tremendous number of separate somatic mutations to account for all those dots.

The Ds element

But when she tried to map them...they wouldn't hold still! From this, she predicted that the two elements were mobile, and could actually change places within the genome and

She also found rare, unusual and unexpected corn kernel phenotypes in the offspring of her corn crosses:

In this example, the presence of Ac causes Ds to break, and the acentric fragment is lost. The result is hemizygosity at all the loci carried away on the lost fragment, allowing recessive phenotypes to be expressed in the cells derived from the single cell in which this breakage occurred early in the corn kernel's development.

Autonomous and Nonautonomous Elements
In plants, there are two types of transposable elements:

1. autonomous elements (these can tranpose by themselves)

2. nonautonomous elements (which need the input (i.e., enzymatic product) of a separate element in order to transpose).

In Rhoades' early study, Dt was that separate element: it supplied the factors promoting the transposition of a gene segment, and insertion of that segment into the pigment gene (A) disrupted the wild type allele's (A₁) function, causing the mutant, unpigmented a₁ phenotype.

Insertion of an autonomous element is unstable, because it can direct its own transposition over and over. The mutation can occur in each generation; the allele produced by the insertion is called a mutable allele because of its instability.

Insertion of a nonautonomous element is stable, because it needs the products of the autonomous element in order to transpose and produce the mutant allele.

Let's look:

And the kicker: Rarely, an Ac type was sometimes found to transform into the Ds type, apparently because the Ac element spontaneously turned into a Ds element. (This could mean that Ds is simply a mutant version of Ac that has lost the ability to encode the elements that allow it to jump around.)

When McClintock first reported her findings in the 1960s, most people believed that this was something unique to corn. But later, as transposable elements were discovered in E. coli, yeast, and higher organisms, it became apparent that she had been the first to describe a phenomenon that was far more universal, suggesting that genomes were far more dynamic than first supposed. In 1983, she was awared the Nobel Prize in Physiology or Medicine for her early work on corn transposons.

Several models have been proposed for transposon insertion mechanism have been proposed. The simplest and most elegant may be that of J. Shapiro. It partly explains the presence of direct and/or inverted repeats where transposons insert.

Insertion Sequences: Prokaryotic Transposable Elements

When an IS appears in any of the three genes of the gal operon (E for epimerase, T for transferase and K for kinase), the normal transcription of the gene is disrupted.

Insertion of an IS affects only the transcription of the genes downstream from the insertion. For example, if the IS occurs late in the E gene, the T and K genes might be disrupted, but the E might not be, and epimerase is still manufactured.

This phenomenon is known as a polar mutation, since there is directionality to the transcriptional effects.

Transposons: More Prokaryotic Mischief

In the 1950's a strain of Shigella bacteria appeared in Japanese hospitals. The normal strains of this bacterium are sensitive to a wide spectrum of antibiotics. But a Shigella strain isolated from patients with a severe dysentery, was discovered to be resistant to most antibiotics.

The multiple-drug resistance phenotype was apparently inherited as a single package--and not only by other Shigella. Other bacterial species could also obtain this resistance.

The problem was a self-replicating episome, a bacterial genetic element capable of

• replicating freely in the cytoplasm or
• being inserted into the bacterial chromosome to replicate along with the chromosome

The R factor is transferred rapidly between bacteria upon conjugation. In the cytoplasm, it exists as a plasmid.

As you may recall, plasmids in bacteria often carry genes that confer resistance to antibiotics

If one denatures the DNA of these R Factors and allows them to slowly renature, portions of the plasmid form a stem loop.

The genes conferring drug resistance are usually located on the LOOP of the stem loop. This is located between two inverted repeat (IR) sequences, which create the stem loop.

The resistance genes in the loop, along with their flanking IR sequences are known as a transposon. The regions between the IR sections are known as the resistance transfer region (RTR), since that's what carries the antibiotic resistance genes.

Two mechanisms for transposition are known in prokaryotes:

1. replicative - the transposon is copied and inserted elsewhere, leaving the original in its place

2. conservative - transposon is excised and reinserted elsewhere.

Both mechanisms generate a repeated sequence of the target DNA (i.e., the DNA in which the transposon is inserted).

Although transposons may excise without affecting surrounding DNA, they often generate a high incidence of deletions in their vicinity. These can consist of part of the element and part of the adjacent DNA.

When varying lengths of the surrounding DNA are excised along with the transposon, imprecise excision is said to have taken place.

When the transposon is excised and deleted portions of the adjacent DNA are restored, precise excision is said to have taken place.

Imprecise excision is far more common than precise excision.

Phage µ

This temperate virus (a bacteriophage) inserts into the genome of E. coli.

If more than one µ is present, they can cause deletions, insertions and translocations of the host's chromosome if both excise at once.

µ replicates with the host c'some, and generally does not form a plasmid.

Transposable Genetic Elements in Other Eukaryotes

• retrotransposon - transposable element in yeast that creates an RNA intermediate (via the activity of reverse transcriptase) to effect transposition (It acts a lot like a retrovirus which--when copied into double-stranded DNA and inserted into the host genome--is called a provirus.)

As much as 10% of Drosophila's genome may consist of families of dispersed, repetitive DNA sequences that move about as discrete units. Three general types are known and named:

• copia-like elements - these are scattered throughout the genome, and code for many of the mRNA's found in Drosophila. (The yellow regions on the accompanying diagram, linked below, are Long direct Terminal Repeats (LTRs). Within the LTR, the target sequence is duplicated--a universal feature of all transposable element insertions.)

  Copia-like elements have properties reminiscent of a provirus.

• FB elements - these contain numerous inverted repeats. Insertion of FB elements has been shown to cause several types of unstable mutations in Drosophila.

• P elements - These have been useful in allowing geneticists to formulate models for the mechanisms of transposition.

Let's have a closer look at P Elements

The Matings:

M female x P male --> dysgenesis

M male x P female --> normal offspring

• A large percentage of dysgenic flies (sterile at high temperatures, but reproductive at normal temperatures) showed evidence that the dysgenic mutations could be easily and frequently reversed, usually in the germline.
• The flies with very high reversion to wild type are generally those with the M cytotype.
• Hypothesis: the mutations are being caused by the insertion of foreign DNA--which could later readily and spontaneously excise, reversing the mutations.
• Probing (with an eye color gene) to recover a particular dysgenic gene revealed that most of the white eye mutations in dysgenic offspring were caused by the insertion of a genetic element into the wild type eye color locus (white eyes resulted from disruption of the wild type red pigment deposition gene).
• This element was named the P element.
• It is present (30-50 copies per genome) in P cytotype flies, but completely absent in M cytotype flies
• The P element can be 0.5 - 2.9 kb in length, but it's always flanked by a 31-base, perfect inverted repeat (red flag for transposable element)
• The complete P element encodes four genes, one of them an enzyme coding for a transposases (what do you suppose this enzyme facilitates?).
• The current hypothesis: The P element encodes not only transposase, but also repressor products that inactivate transposase.
• In P cytotype (wild type) flies, there are many P elements. Thus leads a high concentration of the P element products are produced in P cytotype flies.
• Because there are transposase repressors produced along with the transposase, P elements are immobile: transposase cannot operate on them. No dysgenic mutations occur.
• Because M cytotype (mutant) flies lack the P element, they also they lack the repressor protein products that normally are deposited in the cytoplasm.
• Hence, if the cross of a P cytotype male fly with an M cytotype female fly will yield an embryo with (1) maternal cytoplasm lacking P elements plus (2) a male-donated nucleus which has some inserted P elements.
• In such a hybrid, the P elements will make enough transposase to cause the P element to jump around and cause dysgenesis, but since no repressors have been laid down in the M type maternal cytoplasm, the transposase works fine, causing transposition.
• Apart from being just plain interesting, P elements have become a major tool for the geneticist working on Drosophila genetics, since they are very useful for tagging genes to be cloned and for inserting genes transgenically.

Modern Transposable Genetic Vocabulary

The disrupted gene is said to be nonautonomous. Its expression depends not only on its own existence, but also on the action of the controlling elements (and the presence/absence of the regulator).

A gene that is always potentially turning on and off via the insertion of a receptor element (unpredictable though it might be) is said to be autonomous. In such a gene, it is probable that the regulator gene (Ac, for example) has actually inserted itself into the target gene and does the inactivation (by jumping in) or activation (by jumping out) all by itself.

A World of Eukaryotic TGEs

This may help explain the C-value paradox: There appears to be little correlation between the size of an organism's genome and its biological complexity.

Nearly half of the human genome appears to consist of transposable elements, mostly long interspersed elements (LINEs) and short interspersed elements (SINEs). Most of these can no longer move about, but retain the vestiges of former mobility (e.g., inverted repeats). A vast number also are included only in introns, and are excised and never transcribed. They are evolutionary relics rendered harmless by the points of their insertion and by the host's regulatory mechanisms.

A few elements, however, are still able to move around, and some are known to be responsible for causing human disorders by inserting into specific locations:

This is likely to be only small, initial list. More are undoubtedly going to be found.

In grasses used by humans for grain production, differences in genome size can largely be attributed to different quantities of inserted LTR transposons. Except for the transposon regions, the different grasses show a great deal of synteny in their genomes.

So maybe in the long run, we'll be glad of our little passengers, and they'll eventually be paying their way by means we can't yet foresee.