Tata, 2 yo, Boy CC: Looked Very Pale HT PE LE Looked very pale had for 6 months This similar health happens to both family members of father and mother. Parents are carriers (from screening) bthalassemia mutation in b-globin chain (+)
Tata, 2 yo, Boy CC: Looked Very Pale HT PE LE Looked very pale had for 6 months This similar health happens to both family members of father and mother. Parents are carriers (from screening) bthalassemia mutation in b-globin chain (+)
Tata, 2 yo, Boy CC: Looked Very Pale HT PE LE Looked very pale had for 6 months This similar health happens to both family members of father and mother. Parents are carriers (from screening) bthalassemia mutation in b-globin chain (+)
CC: Looked Very Pale HT PE LE Looked very pale had for 6 months This similar health happens to both family members of father and mother They receive blood transfusion reguraly Immunization (+) Sick (-) Nutritional problem (-) Chronic bleeding (-) Screened abnormalities (-) Looks pale (+) Conjunctiva anemis (+) Sclera yellowish (+) Abdomen hepatosplenomegaly (+)
Haemoglobin (normal) His parents are carriers (from screening) - thalassemia Mutation in -globin chain (+) Diagnosis: -Thalassemia Treatment: Pharmacology: - Non-Pharma: Routine blood transfussion FINAL CONCEPT
, 2 YO Mutation In Gene Expression Parents Carrier Gene Exp & Gene Tech Mutation Failure In mRNA Splicing Nucleic Acid, DNA, RNA, & Protein Syntesys Mutation -Globin Chains Imbalance Of -Globin Chains -Thalassemia Mayor Basic Diagnosys and Clinicall Signs Free -Chains Agregat -Chains Erythroblast Destroyed (Ineffective Erytrhopoesis) Free Precipitates to Damage RBC Abnormal Structure Intravascular Hemolysys Erytrocyte Hb RBC Life Spen Spleen Destroys RBC RBC RBC Production Erythropoetic Tissues (In Liver & Spleen) / Work Hard Pale Anemia Heme
Hepatosplenomegaly Biliverdin Unconjugat ed Bilirubin Icteric Sclera Treatment BHP & PHOP -THALASSEMIA MAYOR 1. Explain the structure, function, resources and utility of nucleic acid 2. Explain the DNA organization, replication and repair 3. Explain the RNA structure, synthesis and processing 4. Explain protein synthesis and genetic code 5. Explain the regulation of gene expression 6. Explain the gene technology and genetic examination for diagnosis 7. Explain the definition of gene, chromosom, and mutation 8. BHP: Ethical issue in frequent blood transfusion 9. PHOP: Premarital screening
NUCLEIC ACID 1. Structure of Nucleic Acid
Each nucleotide is composed of: Pentose sugar o Deoxyribose (DNA) without oxygen atom on carbon-2 o Ribose (RNA)
Nitrogen Bases o Purin (Body : Adenine, Guanine ; Free : Xanthine, Hypoxanthine) o Pyrimidine (Cytocine, Thymine (DNA), Uracil (RNA))
Phosphate group o Provided by H3PO4, able to support segments of nucleic acid
2. Function / Utility Form genetic code important component of protein synthesis Energy source for protein synthesis Gene therapy and gene technology Metabolic process
3. Source : - Amino acids - Sugar - Food rich in nucleotides (brain, spinach, anchovy, caviar) - Nucleotide released in intracellular metabolism
DNA Structure Nucleotide : Consist of 3 major component : pentose sugar, phosphate group, and nitrogen base DNAs structure is double helix that coiled around common axis. The chains are paired in an antiparallel manner, that is, the 5end of one strand is paired with the 3- end of the other strand Complementary chain structure of DNA show that the amount of purine and pyrimidine are the same (A + G = C + T). Adenine will be paired with thymine by 2 hydrogen bond, while Guanine will be paired with Cytosine by 3 hydrogen bond Backbone of DNA consist of phosphate-sugar group. Pentose sugar of DNA is 2- deoxyribose 2 nucleotide joined by phosphodiesther bond on 3rd or 5th C atom Nucleosome : consist of DNA segment that warp around histone octamer. Nucleosome + H1 histone (histone protein tightly bond to chromatin) called chromatosome. Histone protein : protein with + charge that contain may arginine and lysine that could interact with charge and phosphate group p(ionic bond). Kind of histone protein H1 joined in the solenoid H2A H2B H3 H4 There are three major structural forms of DNA: the B form, described by Watson and Crick in 1953, the A form, and the Z form. The B form is a right-handed helix with ten residues per 360 turn of the helix, and with the planes of the bases perpendicular to the helical axis. The A form is produced by moderately dehydrating the B form. It is also a right-handed helix, but there are eleven base pairs per turn. The Z-DNA is a left handed helix that contains about twelve base pairs per turn.
replication Type of DNA replication accepted by universal is semiconservative type. The process are : Separation of the two complementary DNA strands: DNA strain must first separate (or melt), at least in a small region. In prokaryote, it begins at a single, unique nucleotide sequencea site called the origin of replication. In eukaryote, replication begins at multiple sites along the DNA helix. Helicase is the enzyme responsible for separating the double chain on the replication forks on the recognizable sequence of DNA. Single strain DNA protein is responsible to prevent the two separated DNA chain back together Because of the separation of DNA chain in one side, the other part of DNA starts getting tight. Topoisomerase is responsible for unwinding tight parts of DNA. After the DNA chain is separated into 2 part, the synthesis begins. DNA Polymerase 3 starts to synthesis new-born DNA. The synthesis begins from the 3 end. Because DNA consist of 2 anti-parallel backbone, that means they move in different direction. So, there are 2 sites : Leading strain : continuously synthesize from 5 to 3 direction without distraction Lagging strain : Because DNA synthesize need 3-OH end, the synthesize must be initiated by RNA-primase. From that, DNA polymerase 3 start to synthesize newborn DNA. After finished synthesizing 1 DNA fragment, the Okazaki fragment, RNA primase are removed by DNA polymerase 1. Then, they will be joined together by ligase enzyme
DNA repair
Mismatch repair
Base excision repair
Nucleotide excision repair
Double strand break
RNA
A. Definition RNA stands for ribonucleic acid, which is a polymeric molecule made up of one or more nucleotides. A strand of RNA can be thought of as a chain with a nucleotide at each chain link. Each nucleotide is made up of a base (adenine, cytosine, guanine, or uracil), a ribose sugar, and a phosphate.
RNA is divided into four types: a. rRNA b. mRNA c. tRNA d. snRNA
B. Structure
A, G, C, U nitrogenous bases pentose sugar (ribose sugar) P phosphate group
C. Function
D. RNA Synthesis RNA synthesis is the process of transcribing DNA nucleotide sequence information into RNA sequence information. It is catalyzed by a large enzyme called RNA polymerase.
In eukaryotic cells it occurs in the cell nucleus, while in prokaryotic cells, it occurs in the cytoplasm.
There are three steps to this process, which are initiation, elongation, and termination. Illustration to these steps is shown in the picture on the next page.
E. RNA Processing
PROTEIN SYNTHESIS AND GENETIC CODE Protein synthesis is translating genetic code to make amino acids which will bind each other to form polypeptides. The genetic code is a dictionary that identifies the correspondence between a sequence of nucleotide bases and a sequence of amino acids. Each individual word in the code is composed of three nucleotide bases. These genetic words are called codons. Codons are presented in the mRNA language of adenine (A), guanine (G), cytosine (C), and uracil (U). The four nucleotide bases are used to produce the three-base codons. Characteristics of genetic code: 1. Specificity (unambigous), a particular codon always codes for the same amino acid. Example: UUU always codes for phenylalanine
2. Universality, the genetic codes virtually universal, same encoding will occur in every synthesis that happens. An exception occurs in mitochondria, wich a few codons have different meanings. 3. Degeneracy, a given amino acid may have more than one triplet coding for it. For example, arginine is specified by six different codons. Only Met and Trp have just one coding triplet. 4. Nonoverlapping and commaless, the code is read from a fixed starting point as a continuous sequence of bases, taken three at a time. For example, AGCUGGAUACAU is read as AGC/UGG/AUA/CAU without any punctuation between the codons.
PROTEIN SYNTHESIS Components required for translation: 1. Amino acid 2. tRNA 3. aminoacyl-tRNA synthetases 4. mRNA 5. ribosome 6. protein factor 7. ATP and GTP(Guanosine Triphosphate) 8. EIF(Eukaryotic Initiation Factor) = certain compound which become factors that help protein synthesis STEPS 1. INITIATION 1) Dissociation of ribosome into its 40s(small ribosome) and 60s units(large ribosome) 2) Binding of ternary complex (met-tRNA/codon start, GTP, eIF-2) to the 40s ribosomal subunit form preinitiation complex 3) Binding of mRNA to the 40s preinitiation complex to form initiation complex 4) Combination of initiation complex with the 60s ribosomal subunit to form 80s initiation complex 2. ELONGATION Catalyzed by proteins called elongation factors 1) Binding of aminoacyl-tRNA to the A site 2) Peptide bond formation Attachment of growing peptide chain to the tRNA in the A site 3) Translocation Displace the peptidyl tRNA(tRNA that carries amino acid) from A site to P site. Ready for new aminoacyl-tRNA attachment and continuity of elongation process. 3. TERMINATION 1) After multiple cycle of elongation, the stop or terminating codon of mRNA (UAA, UAG, UGA) apperas in A site. 2) Complex contain releasing factor with bound GTP will recognizes stop codon appears in A site. 3) The protein and tRNA will be released from the P site because of hydrolysis 4) When hydrolisis and release, 80s ribosome dissociate into 40s and 60s subunit, which are then recycled 5) mRNA is released from ribosome 6) another recycle can be repeated 7)
GENE EXPRESSION
Influenced by 1. Hormones 2. Heavy metals 3. Chemicals
Regulation a. Positive: Expression of genetic information increased by the presence of specific regulatory element (activator) b. Negative: Expression of genetic information diminished by the presence of specific regulatory element (repressor) Regulation response 1. Type A (prokaryotes): An increased extent of gene expression that is dependent upon the continued presence of the inducing signal. When the signal is removed, the amount of gene expression diminishes to its basal level. 2. Type B (during development of organism): Increased amount of gene that is transient even in the continued presence of the regulatory signal. 2. Type C (during development of differenciated function in a tissue or organ): In response to the regulatory signal, an increased extent of gene expression that persists indefinitely after termination of the signal (the signal acts as a trigger)
Regulation of gene expression Pada prokariot, sebagian besar DNA cetakan dapat dilakukan transkripsi namun untuk eukariot hrus dilakukan remodeling kromatin (modifikasi kromatin) terlebih dahulu guna ditranskripsi. Khusus pada prokariot, DNA cetakan itu berupa operon. APA ITU OPERON? Operon: Gugus gugus cetakan DNA yang diawali dengan promoter, operator dan beberapa gen yang bersebelahan(operon lac). Guna promoter adalah tempat terdapatnya RNA polymerase dan operator berfungsi untuk menyalakan dan mematikan(repressor) dalam proses regulasi.Operator pada prokariot berfungsi sama dengan metilasi DNA pada eukariot.
1. Modifikasi kromatin Gen dalam kromatin sangat terpadatkan biasanya tidak ditranskripsikan. Asetilasi histon tampaknya melonggarkan struktur kromatin dan meningkatkan transkripsi. Metilasi DNA umumnya mengurangi transkripsi. 2. Transkripsi Regulasi inisiasi transkripsi: unsur kontrol DNA mengikat faktor transkripsi spesifik. Penekukan DNA memungkinkan aktivator menyentuh protein di promoter, sehingga menginisiasi transkripsi. Regulasi terkoordinasi. 3. Pemrosesan mRNA Penyambungan RNA alternatif 4. Translasi Inisiasi dari translasi dapat dikontrol melalui regulasi dari faktor inisiasi. 5. Pemrosesan dan degradasi protein. Oleh proteasom 6. Degradasi mRNA. Setiap mRNA memiliki panjang usia yang khas, ditentukan sebagian oleh sekuens pada UTR 5 dan 3
GENE EXAMINATION
RFLP (Restriction Fragment Length Polymorphism) RFLP analysis is used to identify changes in genetic sequences which occur at a site where the restriction enzyme cuts. RFLP is based on the chance of comparing each DNA profile resulted from the cutting of targeted DNA with the restriction enzyme. Steps of RFLP: 1. DNA Isolation The process of isolating the DNA fragments with extraction and lytic method. Usually the process is done with the help of homogenization and usage of additional extraction buffer to prevent DNA from damaging. Separation of DNA from other cell components is done using the centrifuge. 2. Cutting of DNA using the restriction enzyme and electrophoresis gel The result of DNA isolation will then cut using certain restriction enzyme which selected carefully. Every restriction enzyme which place in a suitable environment will recognize and slice the DNA to form the fragments. The fragments will then undergo electrophoresis using the Aragosa gel.
Electrophoresis The standard lab procedure for separating DNA by size (e.g. length in base pairs) for visualization and purification. The principle of the basic categories of analyses used to characterize DNA and RNA: Electrophoretic Separation Movement of DNA or RNA in response to an electric field will be proportional to the molecular weight or length of the molecule. This property is used to characterize the size of nucleic acid fragments by electrophoretic separation.
Hybridization The process of combining two complementary single-stranded DNA or RNA molecules and allowing them to form a double-stranded molecule through base pairing. Hybridization is defined as the interaction between two single-stranded nucleic acid molecules to form a duplex (double-stranded) molecule. Principle: based on the complementary base pairing of their respective sequences. High-stringency conditions high temperature (close to Tm ), low salt, and high formamide will only allow the most perfectly matched duplex structures to remain in a stable helix conformation.
GENE TECHNOLOGY
DNA Cloning DNA cloning is a process of producing the identical DNA from its parental DNA. Vectors are the agent that carries DNA fragment into the living cell in order to increase the amount of DNA fragments. There are a few vectors used within the process: 1. Bacteria a. Plasmid : small circular DNA molecule which have an ability to replicate itself b. Phage : viruses that infected the bacteria c. Cosmides : a larger fragments of DNA which resulted from the combination of plasmid and phage 2. Viral a. Adenovirus b. Retrovirus 3. Artificial Chromosome
DNA Recombinant DNA recombinant is the formation of new genetic combination by inserting a DNA molecule into certain vector, making it possible for them to integrate and undergo a replication process within other organism. Purpose of DNA recombinant: 1. Gene mapping: determine the specific location of gene in particular chromosome. 2. Production of protein for research and diagnosis purposes. 3. Examination of molecular analysis in disease.
Gene Therapy Gene therapy is the use of DNA as a drug to treat disease by delivering therapeutic DNA into a patient's cells. Mostly involves using DNA that encodes a functional, therapeutic gene to replace a mutated gene. There are two types of gene therapy, yet only one of which has been used in humans: 1. Somatic Gene Therapy 2. Germline Gene Therapy Principle of gene therapy Inserting precursor cell to the location of the damaged cell so that the precursor cell will have the same expression with damaged cells and able to replace the function of the damaged cells. PCR (Polymerase Chain Reaction) Technic used in certain DNA fragments amplification in an in vitro medium using a pair of oligonucleotide.
Seperating the double-strand DNA, breaking the hydrogen bond in order to form the single- strand DNA. Temperature needed: 92-94 o C. Denaturation Primer forward and reverse is searching for its pair in the DNA strands. Annealing Taq polymerase replicate the DNA fragment. Temperature needed: 72 0 C Elongation MUTATION Definition A mutation is defined as a permanent change in the DNA. Mutatations that affect germ cells are transmitted to the progeny and can give rise to inherited disease. Mutations that arise in somatic cells understandably do not cause hereditary diseases but are important in the genesis of cancers and some congenital malformations. Human genetic disorders can be broadly classified into three categories : 1. Disorders related to mutations in single genes with large effects: Highly penetrant, they usually follow the classic Mendelian pattern of inheritance and are also referred to as Mendelian disorders. 2. Chromosomal disorder: Uncommon, but with high penetrance 3. Complex multigenic disorders: Often expressed in polymorphisms, although it is often of small effect and low penetrance unless in huge amounts of polymorphisms expressed (in multigenic or polygenic conditions as it is termed) Penetrance = the frequency of manifestation of a hereditary condition in individuals. Mutations may result in partial or complete deletion of a gene or affect a single base.
Types of mutations 1. Point mutations It may alter the code in a triplet of bases and lead to the replacement of one amino acid by another in the gene product. There are several types of this mutation . Missense mutations: Replacement of normal amino acid with a very different one that can cause some physiological or physical changes (often detrimental). Eg. -thalassemia due to CTC changed in CAC. . Nonsense mutations: May cause severe translation errors, causing often severe physiological or physical effects. Eg. Beta-thalassemia major due to codon for glutamine (CAG) creates stop codon (UAG), this creates premature termination of betaglobin gene translation and causes severe form of anemia called o- thalassemia. . Silent mutation: No change in protein function can be observed due to the mutation being silent (has no visible physiological or physical effects on the mutant subject). Caused due to base substitution, there are 2 types of base substitution : . Transition: Changes purine to purine / pyrimidine to pyrimidine. . Transversion: Changes purine to pyrimidine / pyrimidine to purine. 2. F rameshift mutations It alters the reading frame of the DNA strand, it is usually detrimental for an organisms fitness. They are caused by : . Insertions: Addition of one or more DNA that alters the reading frame of the DNA.
. Deletions: Deletion of one or more DNA that alters the reading frame of the DNA.
3. Trinucleotide-repeat mutations Special category of genetic anomaly. Characterized by amplification (gene duplication) of a sequence of three nucleotides. 4. Chromosomal mutation There are 4 types of chromosome according to the position of centromeres : . Metacentric = at the centre of the chromosome . Submetacentric = near the centre of the chromosome . Acrocentric = near the end of the chromosome . Telocentric = at the end of the chromosome Chromosme mutation is usually more severe than point mutations, there are several types of chromosmal mutations : . Deletion . Duplication . Inversion
BHP & PHOP BHP Informing patient about the reason why the patient should get the transfusion blood. Informing patient about the procedure of blood transfussion. Informing patient about the risk, benefit, purpose, and requirement of blood transfusion to the donature and the recepient.
PHOP Preventive : Doing genetic counseling and genetic screening before married (premartial screening ) Promotive : Educate and persuading people the importance of genetic counseling before married to prevent the genetical disease happen Currative : Giving the patient blood transfusion, stem cell transfusion, and bone marrow transplant. Rehabilitative : Doing routine check up to prevent any risk happen after blood transfusion