I-CreI
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I-CreI is a
homing endonuclease The homing endonucleases are a collection of endonucleases encoded either as freestanding genes within introns, as fusions with host proteins, or as self-splicing inteins. They catalyze the hydrolysis of genomic DNA within the cells that synthes ...
whose
gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
was first discovered in the
chloroplast A chloroplast () is a type of membrane-bound organelle, organelle known as a plastid that conducts photosynthesis mostly in plant cell, plant and algae, algal cells. Chloroplasts have a high concentration of chlorophyll pigments which captur ...
genome of ''
Chlamydomonas reinhardtii ''Chlamydomonas reinhardtii'' is a single-cell green alga about 10 micrometres in diameter that swims with two flagella. It has a cell wall made of hydroxyproline-rich glycoproteins, a large cup-shaped chloroplast, a large pyrenoid, and a ...
'', a species of unicellular
green algae The green algae (: green alga) are a group of chlorophyll-containing autotrophic eukaryotes consisting of the phylum Prasinodermophyta and its unnamed sister group that contains the Chlorophyta and Charophyta/ Streptophyta. The land plants ...
. It is named for the facts that: it resides in an Intron; it was isolated from ''Clamydomonas reinhardtii''; it was the first (I) such gene isolated from ''C. reinhardtii''. Its gene resides in a group I
intron An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word ''intron'' is derived from the term ''intragenic region'', i.e., a region inside a gene."The notion of the cistron .e., gen ...
in the 23S
ribosomal RNA Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosomal ...
gene of the ''C. reinhardtii'' chloroplast, and I-CreI is only expressed when its
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
is spliced from the
primary transcript A primary transcript is the single-stranded ribonucleic acid (RNA) product synthesized by transcription of DNA, and processed to yield various mature RNA products such as mRNAs, tRNAs, and rRNAs. The primary transcripts designated to be mRNA ...
of the 23S gene. I-CreI
enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
, which functions as a
homodimer In biochemistry, a protein dimer is a macromolecular complex or protein multimer, multimer formed by two protein monomers, or single proteins, which are usually Non-covalent interaction, non-covalently bound. Many macromolecules, such as proteins ...
, recognizes a 22-nucleotide sequence of duplex DNA and cleaves one phosphodiester bond on each strand at specific positions. I-CreI is a member of the LAGLIDADG family of homing endonucleases, all of which have a conserved LAGLIDADG amino acid motif that contributes to their associative domains and active sites. When the I-CreI-containing intron encounters a 23S allele lacking the intron, I-CreI enzyme "homes" in on the "intron-minus" allele of 23S and effects its parent intron's insertion into the intron-minus allele. Introns with this behavior are called mobile introns. Because I-CreI provides for its own propagation while conferring no benefit on its host, it is an example of
selfish DNA Selfish genetic elements (historically also referred to as selfish genes, ultra-selfish genes, selfish DNA, parasitic DNA and genomic outlaws) are genetic segments that can enhance their own transmission at the expense of other genes in the genome, ...
.


Discovery

I-CreI was first observed as an intervening sequence in the 23S
rRNA Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosomal ...
gene of the ''C. reinhardtii'' chloroplast genome. The 23S gene is an
RNA gene A non-coding RNA (ncRNA) is a functional RNA molecule that is not translated into a protein. The DNA sequence from which a functional non-coding RNA is transcribed is often called an RNA gene. Abundant and functionally important types of non-co ...
, meaning that its transcript is not translated into protein. As RNA, it forms part of the large subunit of the
ribosome Ribosomes () are molecular machine, macromolecular machines, found within all cell (biology), cells, that perform Translation (biology), biological protein synthesis (messenger RNA translation). Ribosomes link amino acids together in the order s ...
. An
open reading frame In molecular biology, reading frames are defined as spans of DNA sequence between the start and stop codons. Usually, this is considered within a studied region of a prokaryotic DNA sequence, where only one of the six possible reading frames ...
coding for a 163-amino acid protein was found in this 23S intron, suggesting that a protein might facilitate the homing behavior of the mobile intron. Furthermore, the predicted protein had a LAGLIDADG motif, a conserved amino acid sequence that is present in other proteins coded for in group I mobile introns. A 1991 study established that the ORF codes for a DNA endonuclease, I-CreI, which selectively cuts a site corresponding to where the intron is spliced out of the 23S primary transcript. The study also showed that the intron was able to invade 23S alleles that did not already have it.


Mechanism of propagation

I-CreI has evolved to cut a 22-nucleotide sequence of DNA that occurs in
allele An allele is a variant of the sequence of nucleotides at a particular location, or Locus (genetics), locus, on a DNA molecule. Alleles can differ at a single position through Single-nucleotide polymorphism, single nucleotide polymorphisms (SNP), ...
s of the 23S ribosomal RNA gene that lack the I-CreI-containing intron. When such an "intron-minus" allele is cut, pathways of double-strand break repair are activated in the cell. The cell uses as a template for repair the 23S allele that yielded the responsible I-CreI enzyme, thus replicating the I-CreI-containing intron. The resulting "intron-plus" allele no longer contains an intact homing site for the I-CreI enzyme, and is therefore not cleaved. Since this intron provides for its own replication without conferring any benefit on its host, I-CreI is a form of
selfish DNA Selfish genetic elements (historically also referred to as selfish genes, ultra-selfish genes, selfish DNA, parasitic DNA and genomic outlaws) are genetic segments that can enhance their own transmission at the expense of other genes in the genome, ...
.


Structural studies and possible applications

Because I-CreI has evolved to cut such a long sequence of DNA, unlike
restriction endonucleases A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
that typically cut four- or six-nucleotide sequences, it is capable of cutting a single site within a very large
genome A genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding genes, other functional regions of the genome such as ...
. A four- or six-nucleotide sequence is expected to occur many, many times in a genome of millions or billions of nucleotides simply by chance, whereas a 22-nucleotide sequence might occur only once (109/46 vs. 109/422). This specificity of I-CreI cleavage makes I-CreI a promising tool for
gene targeting Gene targeting is a biotechnological tool used to change the DNA sequence of an organism (hence it is a form of Genome Editing). It is based on the natural DNA-repair mechanism of Homology Directed Repair (HDR), including Homologous Recombinat ...
. If a person were to have a disease due to a defective
allele An allele is a variant of the sequence of nucleotides at a particular location, or Locus (genetics), locus, on a DNA molecule. Alleles can differ at a single position through Single-nucleotide polymorphism, single nucleotide polymorphisms (SNP), ...
of some
gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
, it would be helpful to be able to replace that allele with a functional one. If one could cause I-CreI to cut the DNA only in the defective allele while simultaneously providing a normal allele for the cell to use as a repair template, the patient's own
homologous recombination Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in Cell (biology), cellular organi ...
machinery could insert the desired allele in place of the dysfunctional one. The specificity of I-CreI also allows for the reduction of deleterious effects due to
double-strand breaks DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. A weakened capacity for DNA repair is a risk factor for the development of cancer. DNA is constantly modified ...
outside of the gene of interest. In order to use I-CreI as a tool in this fashion, it is necessary to make it recognize and cleave sequences of DNA different from its native homing site. An ''
Escherichia coli ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Escherichia'' that is commonly fo ...
'' genetic system for studying the relationship between I-CreI structure and its homing site specificity was created in 1997. In 1997, the structure of the I-CreI protein was determined, and in 1998, its crystal structure bound to its native DNA homing site was solved, greatly aiding research in altering the homing site recognition of the protein. Mutant forms of the protein have since been created that exhibit altered homing site specificity. A genetic system in ''
Saccharomyces cerevisiae ''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungal microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have be ...
'' has also been created, yielding additional I-CreI mutants with modified homing site specificities. I-CreI has already been used successfully to induce homologous recombination in ''
Drosophila melanogaster ''Drosophila melanogaster'' is a species of fly (an insect of the Order (biology), order Diptera) in the family Drosophilidae. The species is often referred to as the fruit fly or lesser fruit fly, or less commonly the "vinegar fly", "pomace fly" ...
'', an extremely popular
eukaryotic The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
model organism A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model organism will provide insight into the workings of other organisms. Mo ...
. It seems very likely that advances in molecular biological techniques and generation of a library of I-CreI-derived novel endonucleases will eventually allow for the targeting of many genes of etiological significance.


References

{{reflist Biotechnology Restriction enzymes