Jorge GarcíaIan López

Carlos Ugalde

Tazswana's Story: How Alternative Splicing Leads to Genetic Disease

and Cure

Handout 1—“Junk DNA”Junk DNA- are the introns

of a pre mRNA.They are junk because of

human evolution.In prokaryotes splicing

does not occur.Splicesome is the

machinery in charge of cutting and pasting mRNA so a person cannot have a disease caused by having noncoding regions in their RNA.

Handout 2—β-Globin Inheritance Pattern

Tazswana’s Story

Handout 4—Tazswana’s Blood Test

Miss X’s Mother’s Sample

Normal Sample

Miss X’s Sample

The subject has severe symptoms since the function of hemoglobin is to transport oxygen to the body for aerobic respiration, and her red blood cells, are small and weak due to the color and distortion of shape. Compared to the mother and a normal person, the altered shape causes a protein to function improperly or not at all making her symptoms severe.

Handout 4—Tazswana’s Blood Test

Handout 6—Tazswana’s RNA Test

Handout 7—Analyzing Tazswana’s Intron

Handout 8—Pre-mRNA Gene Therapy for Tazswana

Alternative Gene Splicing

Post-transcriptional modification in which a single gene can code for multiple proteins.

Done in eukaryotes, prior to mRNA translation, by the differential inclusion or exclusion of regions of pre-mRNA.

Enables a single gene to increase its coding capacity, allowing the synthesis of protein isoforms that are structurally and functionally distinct.

Modes of Gene Splicing Alternative selection of promoters: this is the only method of splicing which

can produce an alternative N-terminus domain in proteins. In this case, different sets of promoters can be spliced with certain sets of other exons.

Alternative selection of cleavage/polyadenylation sites: this is the only method of splicing which can produce an alternative C-terminus domain in proteins. In this case, different sets of polyadenylation sites can be spliced with the other exons.

Intron retaining mode: in this case, instead of splicing out an intron, the intron is retained in the mRNA transcript. However, the intron must be properly encoding for amino acids. The intron's code must be properly expressible, otherwise a stop codon or a shift in the reading frame will cause the protein to be non-functional.

Exon cassette mode: in this case, certain exons are spliced out to alter the sequence of amino acids in the expressed protein.

ImportanceNew proteins could be allowed to evolve much faster

than in prokaryotes.Invalidates the old theory of one DNA sequence

coding for one polypeptide (the "one-gene-one-protein" hypothesis)

For eukaryotes it was a very important step towards higher efficiency, because information can be stored much more economically.

Gene Therapy Gene therapy is a technique for correcting defective genes responsible

for disease development. A normal gene may be inserted into a nonspecific location within the

genome to replace a nonfunctional gene. This approach is most common.

Procedure:A normal gene is inserted into the genome to replace an abnormal,

disease-causing gene. A carrier molecule called a vector must be used to deliver the

therapeutic gene to the patient's target cells. The most common vector is a virus that has been genetically altered to carry normal human DNA (retrovirus).

Target cells such as the patient's are infected with the viral vector.The vector then unloads its genetic material containing the

therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state.

Gene Therapy The commonly used virus in gene therapy is called a

retrovirus. It is a class of viruses that can create double-stranded DNA copies of their RNA genomes. Human immunodeficiency virus (HIV) is a retrovirus.

The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA.

Current gene therapy is experimental and has not proven very successful in clinical trials.

Factors that have kept the therapy from becoming an effective treatment:Short-lived nature of gene therapy Immune response Problems with viral vectors Multigene disorders

Gene TherapyIn the case of

thalassemia, it is required the transfer of healthy genes into the stem cells of the bone marrow.

However, an important problem with the retroviruses methods is ensuring that the β-chains produced as a result of the healthy gene introduced are of sufficiently high quantity and quality over the long term.

Bibliographies• Premier Biosoft International, (2008). Gene

Splicing Overview and Techniques. Retrieved April 27, 2008, from Premier Biosoft International Web site: http://www.premierbiosoft.com/tech_notes/gene- splicing.html

• Genetics Home Reference, (2008, April 25). What is gene therapy?. Retrieved April 28, 2008, from Genetics Home Reference Web site: http://ghr.nlm.nih.gov/handbook/therapy/genetherapy