Recombinant DNA technology
Genes are DNA sequences that code for protein. Gene splicing is a form of genetic engineering where specific genes or gene sequences are inserted into the genome of a different organism. Gene splicing can also specifically refer to a step during the processing of deoxyribonucleic acid (DNA) to prepare it to be translated into protein.
Gene splicing can also be applied to molecular biology techniques that are aimed at integrating various DNA sequences or gene into the DNA of cells. Individual genes encode specific proteins and, based on the outcome of the Human Genome Project, it is estimated that there are approximately 30,000 genes in each cell of the human body. Because the cellular functions in different tissues have varying purposes, the genes undergo a complex concerted effort to maintain the appropriate level of gene expression in a tissue specific manner. For example, muscle cells require specific proteins to function, and these proteins differ remarkably from proteins in brain cells. Although the genetic information is, for the most part, the same in both cell types, the different functional purposes result in different cellular needs and therefore different proteins are produced in different tissue types.
Genes are not expressed without the proper signals. Many genes can remain inactive. With the appropriate stimulation of gene expression, the cell can produce various proteins. The DNA must first be processed into a form that other molecules in the cell can recognize and translate it into the appropriate protein. Before DNA can be converted into protein, it must be transcribed into ribonucleic acid (RNA). There are three steps in RNA maturation; splicing, capping, and polyadenylating. Each of these steps are involved in preparing the newly created RNA, called the RNA transcript, so that it can exit the nucleus without being degraded. In terms of gene expression, the splicing of RNA is the step where gene splicing occurs in this context at specific locations throughout the gene. The areas of the gene that are spliced out are represent noncoding regions that are intervening sequences also known as introns. The DNA that remains in the processed RNA is referred to as the coding regions and each coding regions of the gene are known as exons. Therefore, introns are intervening sequences between exons and gene splicing entails the excision of introns and the joining together of exons. Hence, the final sequence will be shorter than the original coding gene or DNA sequence.
In order to appreciate the role splicing plays in how genes are expressed, it is important to understand how a gene changes into its functional form. Initially, RNA is called precursor RNA (or pre-RNA). Pre-RNAs are then further modified to other RNAs called transfer RNA (tRNA), ribosomal RNA (rRNA), or messenger RNA (mRNA). mRNAs encode proteins in a process called translation, while the other RNAs are important for helping the mRNA be translated into protein. RNA splicing creates functional RNA molecules from the pre-RNAs.
Splicing usually proceeds in a predetermined way for each gene. Experiments which have halted transcript formation at different intervals of time show that splicing will follow a major pathway beginning with some intron and proceeding selectively to another, not necessarily adjacent, intron. Although other pathways can be followed, each transcript has its own primary sequence for intron excision.
A single gene can be processed to create numerous gene products, or proteins and this process is referred to as alternative splicing. In this case, a different combination of exons remains in the processed RNA. Alternate gene splicing at various intron-exon sites within a gene can be used to create several proteins from the same pre-RNA molecule. Proteins are made up of multiple domains. Different exons can code for different domains. Selective splicing can remove unwanted exons as well as introns. The combinations of proteins that can be produced from alternate splicing are related in structure or function but are not identical. By using a single gene to create multiple proteins, the cells DNA can be utilized more efficiently.
Alternate splicing can be tissue specific such that different proteins are made from the same original gene by two or more different cell types. Or one cell type may make multiple configurations using the same gene. For instance, a type of immune cell called a B-cell manufactures antibodies to numerous antigens. Antigens are foreign substances, which trigger immune responses and antibodies bind and antigens so that they can be broken down and removed. Although an infinite number of antibodies can be produced, all antibodies fall into one of five basic subtypes. Alternate splicing is used to create these five antibody-types from the same gene.
Antibodies are made up of multiple immunoglo-bulin (Ig) molecules. These molecules in turn have multiple domains. A particular domain called the heavy chain constant region distinguishes the five antibody subtypes, called IgM, IgD, IgG, IgE, and IgA. The different types of antibodies serve various functions in the body and act in distinct body tissues. For example, IgAs are secreted into the gastrointestinal mucosa, and IgGs passes through the placenta. The gene encoding these heavy chain regions contains exons that direct the production of individual subtypes, and the gene is alternately spliced to yield a final mRNA transcript, which can make any one of them.
Most genes yield only one transcript; however, genes that yield multiple transcripts have numerous cellular and developmental roles. Alternate splicing controls sex determination in Drosophila melanogaster flies. And a number of proteins are differentially expressed from the same gene in various cells. Different muscle cells use alternate splicing to create cell-specific myosin proteins. And embryonic cells in varying developmental stages produce multiple forms of the protein, retinoic acid. Some transcripts differ from related transcripts in the 5’ end and others can vary at the 3’ end.
The molecules or molecular complexes that actually splice RNA in the cellular nucleus are called spliceosomes. Spliceosomes are made of small sequences of RNAs bound by additional small proteins. This spliceosome complex recognizes particular nucleotide sequences at the intron-exon boundary. DNA and RNA are both generally read in the 5’ to 3’ direction. This designation is made on the basis of the phospho-diester bonds, which make up the backbone of DNA and RNA strands. Introns are first cut at their 5’ end and then at their 3’ end. The two adjacent exons are then bonded together without the intron. The spliceosome is an enzymatic complex which performs each of these steps along the pre-RNA to remove introns.
The small RNAs which make up the spliceosome are not mRNAs, rRNAs, or tRNAs; they are small nuclear RNAs (snRNA’s). snRNAs are present in very low concentrations in the nucleus. The snRNAs combine with proteins to comprise, small nuclear ribo-nuclearprotein particles. Several snRNPs aggregate to form a spliceosome. This secondary structure recognizes several key regions in the intron and at the intron-exon border. In essence, snRNPs play a catalytic splicing role. The absence of individual snRNP components can inhibit splicing. snRNPs are only one of many complexes which can regulate gene expression.
In addition to snRNPs, some introns have auto (self) splicing capabilities. These introns are called group II introns. Group II introns are found in some mitochondrial genes, which come from a genome that is separate from the nucleus and is located in small compartments within the cell called mitochondria. Mitochrondria functions to provide energy for the cells energy requirements. Although all chromosomal DNA is located in the nucleus, a few genes are located in the cells mitochondria. Group II introns form secondary structures using their internal intron region in a similar way to nuclear introns. However, these mitochondrial introns direct exon-exon rejoining by themselves without snRNPs.
Various splicing signal sequences are universal and are found within every intron site spliced, while some signal sequences are unique to individual genes. DNA is made up of bases called nucleotides, which represent the DNA alphabet. There are four bases, Adenine (A), Guanine (G), Thymine (T), and Cytosine (C). Most introns in higher life forms begin with the nucleotide sequence G-T and end with the sequence A-G. The sequences define the “left” (5’) and “right” (3’) borders of the intron and are described as conforming to the GT-AG rule. Mutations in any of these four positions produce introns that cannot be removed by normal splicing mechanisms. Within the intron is another highly conserved sequence that has some variability in the genes of a species; this region (called the branch site) is the area that connects to the 5’ end of the intron as it is cut and then curls around to form a lariat shape. This lariat is a loop in the intron which is formed as it is removed from the maturing RNA.
Splicing can also involve molecules other than mRNA. tRNAs, which play a crucial role of aligning amino acids along a protein being synthesized can undergo splicing. tRNAs are encoded by DNA just
Antibody —A molecule created by the immune system in response to the presence of an antigen (a foreign substance or particle). It marks foreign microorganisms in the body for destruction by other immune cells.
Antigen —A molecule, usually a protein, that the body identifies as foreign and toward which it directs an immune response.
Capping —A modification to the 5’ end of a mature mRNA transcript.
Cytoplasm —All the protoplasm in a living cell that is located outside of the nucleus, as distinguished from nucleoplasm, which is the protoplasm in the nucleus.
Deoxyribonucleic acid (DNA) —The genetic material in a cell.
Exons —The regions of DNA that code for a protein or form tRNA or mRNA.
Gene —A discrete unit of inheritance, represented by a portion of DNA located on a chromosome. The gene is a code for the production of a specific kind of protein or RNA molecule, and therefore for a specific inherited characteristic.
Genome —The complete set of genes an organism carries.
Introns —Noncoding sequences in a gene that are spliced out during RNA processing.
Mitochondria —Intracellular organelle that is separate from the nucleus, has it’s own genome and is important for producing energy for various tissues.
Polyadenylation —A modification to the 3’ end of a mature mRNA transcript.
Recombinant DNA —DNA that is cut using specific enzymes so that a gene or DNA sequence can be inserted.
Splicesome —The intracellular machinery that processes RNA by removing introns from the sequence.
like all other RNA molecules. However, tRNAs have a unique structure and function distinct from other RNA molecules in that they are responsible for matching the actual protein building blocks (amino acids) from the encoded nucleotide sequence to build a protein, or polypeptide. Since these specialized RNAs have unique conformations, enzymes that join exons after intron removal differ from those that join introns in other RNA molecules. While introns are removed, and exons are joined, the enzymatic molecules are not the same as those used for mRNA processing. Intron removal in tRNA processing is less dependent on internal intron sequences compared to other RNA introns.
Advances in understanding the mechanisms that describe how gene splicing occurs has lead to the ability of scientists to cut and anneal nucleotide sequences, also called recombinant DNA technology. Since splice literally means the joining of separate ends, gene splicing refers to the joining of almost any nucleotide sequences to create a new gene product or to introduce a new gene sequence. Hence, just about any genetic sequence could be spliced into another sequence.
Certain enzymes called restriction enzymes are used in laboratories to splice, connect (or ligate), and remove or add nucleotides to sequences. Restriction enzymes are used in recombinant DNA technology to remove and insert genetic sequences from and into other sequences. This technology has enabled some biotechnology and pharmaceutical companies to manufacture large quantities of essential proteins for medical and research purposes. For example, a human insulin protein can be made in great supply by inserting the insulin gene into the genome of bacteria, for example, in order to produce large amounts of the protein. Like a photocopy machine, such sequences can produce lots of insulin for diabetics who are not able to make enough insulin on their own. These patients can then self-inject the purified insulin to treat their disease.
Using gene-splicing technology, vaccines have been produced. DNA from a virus can be spliced into the genome of a harmless strain of bacterial strain. When the bacteria produced the viral protein, this protein can be harvested. Since bacteria grow quickly and easily, a large amount of this protein can be extracted, purified and used as a vaccine. It is introduced into an individual by injection, which will elicit an immune response. When a person is infected with a virus by natural exposure, a rapid immune response can be initiated due to the initial inoculation. Another application of gene spicing technology is related to the gene involved in vitamin B production. This gene has been removed from a carrots genome and spliced into the genome of rice. The genetically engineered recombinant rice strain therefore, is modified to produce vitamin B. This can have many health-related benefits, particularly in third world countries that rely on rice as a major food source and do not have access to food sources rich in vitamins.
Gene splicing technology, therefore, allows researchers to insert new genes into the existing genetic material of an organisms genome so that entire traits, from disease resistance to vitamins, and can be copied from one organism and transferred another.
Keller, Evelyn Fox. The Century of the Gene. Boston: Harvard University Press, 2002.
Lambrecht, Bill. Dinner at the New Gene Cafe: How Genetic Engineering is Changing What We Eat, How We Live, and the Global Politics of Food. New York: St. Martin’s Press, 2002.