MOLECULAR BIOLOGY introduction for lab.pptx

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1. MOLECULER BIOLOGY BY MBUYE GILBERT Y MED. LAB SCIENTIST BY Mbuye Gilbert 1

2. This course will introduce students to the principles of molecular biological techniques, especially in the diagnosis of diseases These diseases will range from bacterial, viral and parasitic At the end of the course the students will be able to; Apply the knowledge accrued in performing assays like PCR, paternity testing as well as gain mastery in genes disorders and gene therapy . BY Mbuye Gilbert 2 OBJECTIVES

3. Introduction Mendel Laws, sex chromosomes and sex linkage, chromosome mapping The role of genes in development. DNA replication and transcription The genetic code, mutation, mutagenesis and repair. Protein synthesis (bacteria and Eukaryotes), Gene cloning and: restriction endonucleases, Cloning vectors and gene expression system Construction of genomic and cDNA libraries Characterization of cloned DNA Hybridization, northern and southern blots Gene amplification: PCR DNA sequencing techniques BY Mbuye Gilbert 3 TABLE OF CONTENTS

4. Genetics is the study of how genes bring about characteristics, or traits, in living things and how those characteristics are inherited Genes are specific sequences of nucleotides that code for particular proteins Through the processes of meiosis and sexual reproduction, genes are transmitted from one generation to the next. Augustinian monk Gregor Mendel developed the science of genetics. Mendel performed his experiments in the 1860s and 1870s B ut the scientific community did not accept his work until early in the twentieth century Because the principles established by Mendel form the basis for genetics, the science is often referred to as Mendelian genetics It is also called classical genetics to distinguish it from another branch of biology known as molecular genetics. BY Mbuye Gilbert 4 Introduction

5. Mendel believed that factors pass from parents to their offspring, but he did not know of the existence of DNA. Modern scientists accept that genes are composed of segments of DNA molecules that control discrete hereditary characteristics. Most complex organisms have cells that are diploid Diploid cells have a double set of chromosomes, one from each parent For example, human cells have a double set of chromosomes consisting of 23 pairs, or a total of 46 chromosomes In a diploid cell, there are two genes for each characteristic In preparation for sexual reproduction, the diploid number of chromosomes is reduced to a haploid number. That is, diploid cells are reduced to cells that have a single set of chromosomes. These haploid cells are gametes, or sex cells, and they are formed through meiosis. When gametes come together in sexual reproduction, the diploid condition is reestablished. BY Mbuye Gilbert 5 Introduction

6. The offspring of sexual reproduction obtain one gene of each type from each parent The different forms of a gene are called alleles. In humans, for instance, there are two alleles for earlobe construction One allele is for earlobes that are attached, while the other allele is for earlobes that hang free The type of earlobe a person has is determined by the alleles inherited from the parents. The set of all genes that specify an organism’s traits is known as the organism’s genome . The genome for a human cell consists of about 20,000-30,000 genes The gene composition of a living organism is its genotype . BY Mbuye Gilbert 6 Continuation

7. For a person’s earlobe shape, the genotype may consist of two alleles for attached earlobes, or two alleles for free earlobes, or one allele for attached earlobes and one allele for free earlobes. The expression of the genes is referred to as the phenotype of a living thing. If a person has attached earlobes, the phenotype is “attached earlobes .” If the person has free earlobes, the phenotype is “free earlobes.” Even though three genotypes for earlobe shape are possible, only two phenotypes (attached earlobes and free earlobes) are possible The two paired alleles in an organism’s genotype may be identical, or they may be different An organism’s condition is said to be homozygous when two identical alleles are present for a particular characteristic In contrast, the condition is said to be heterozygous when two different alleles are present for a particular characteristic BY Mbuye Gilbert 7 Continuation

8. In a homozygous individual, the alleles express themselves In a heterozygous individual, the alleles may interact with one another, and in many cases, only one allele is expressed. When one allele expresses itself and the other does not, the one expressing itself is the dominant allele The “overshadowed” allele is the recessive allele. In humans, the allele for free earlobes is the dominant allele If this allele is present with the allele for attached earlobes, the allele for free earlobes expresses itself, and the phenotype of the individual is “free earlobes .” Dominant alleles always express themselves, while recessive alleles express themselves only when two recessive alleles exist together in an individual . Thus , a person having free earlobes can have one dominant allele or two dominant alleles, while a person having attached earlobes must have two recessive alleles. BY Mbuye Gilbert 8 continuation

9. Mendel was the first scientist to develop a method for predicting the outcome of inheritance patterns. He performed his work with pea plants, studying seven traits: plant height, pod shape, pod color, seed shape, seed color, flower color, and flower location Pea plants pollinate themselves. Therefore , over many generations, pea plants develop individuals that are homozygous for particular characteristics. These populations are known as pure lines . In his work, Mendel took pure-line pea plants and cross-pollinated them with other pure-line pea plants. He called these plants the parent generation BY Mbuye Gilbert 9 Inheritance Patterns

10. When Mendel crossed pure-line tall plants with pure-line short plants, he discovered that all the plants resulting from this cross were tall He called this generation the F1 generation (first filial generation) Next, Mendel crossed the offspring of the F1 generation tall plants among themselves to produce a new generation called the F2 generation (second filial generation) Among the plants in this generation, Mendel observed that three-fourths of the plants were tall and one-fourth of the plants were short. BY Mbuye Gilbert 10 Inheritance Patterns

11. Mendel conducted similar experiments with the other pea plant traits Over many years, he formulated several principles that are known today as Mendel’s laws of genetics 1. Mendel’s law of dominance : When an organism has two different alleles for a trait, one allele dominates. 2 . Mendel’s law of segregation : During gamete formation by a diploid organism, the pair of alleles for a particular trait separate, or segregate, during the formation of gametes (as in meiosis). 3.Mendel’s law of independent assortment : The members of a gene pair separate from one another independent of the members of other gene pairs. These separations occur in the formation of gametes during meiosis BY Mbuye Gilbert 11 Mendel’s laws of genetics

12. An advantage of genetics is that scientists can predict the probability of inherited traits in offspring by performing a genetic cross (also called a Mendelian cross) To predict the possibility of an individual trait, several steps are followed First , a symbol is designated for each allele in the gene pair. The dominant allele is represented by a capital letter and the recessive allele by the corresponding lowercase letter, such as E for free earlobes and e for attached earlobes For a homozygous dominant individual, the genotype would be EE For a heterozygous individual, the genotype would be Ee F or a homozygous recessive individual, the genotype would be ee BY Mbuye Gilbert 12 Mendelian crosses

13. The next step in performing a genetic cross is determining the genotypes of the parents and the genotype of the gametes A heterozygous male and a heterozygous female to be crossed have the genotypes of Ee and Ee During meiosis, the allele pairs separate . A sperm cell contains either an E or an e, while the egg cell also contains either an E or an e. BY Mbuye Gilbert 13 Continuation

14. Mendel’s studies have provided scientists with the basis for mathematically predicting the probabilities of genotypes and phenotypes in the offspring of a genetic cross But not all genetic observations can be explained and predicted based on Mendelian genetics Other complex and distinct genetic phenomena may also occur Several complex genetic concepts, described in this section, explain such distinct genetic phenomena as blood types and skin color BY Mbuye Gilbert 14 Principles of Genetics

15. In some allele combinations, dominance does not exist Instead , the two characteristics are equally expressed For instance, snapdragon flowers display incomplete dominance in their color There are two alleles for flower color: one for white and one for red When two alleles for white are present, the plant displays white flowers When two alleles for red are present, the plant has red flowers But when one allele for red is present with one allele for white, the color of the snapdragons is pink. However, if two pink snapdragons are crossed, the phenotype ratio of the offspring is one red, two pink, and one white These results show that the genes themselves remain independent; only the expressions of the genes appear to “blend .” If the gene for red and the gene for white actually blended, pure red and pure white snapdragons could not appear in the offspring BY Mbuye Gilbert 15 Incomplete dominance

16. In certain cases, more than two alleles exist for a particular characteristic Even though an individual has only two alleles, additional alleles may be present in the population This condition is known as multiple alleles An example of multiple alleles occurs in blood type In humans, blood groups are determined by a single gene with three possible alleles: A, B, or O. Red blood cells can contain two antigens, A and B The presence or absence of these antigens results in four blood types: A, B, AB, and O If a person’s red blood cells have antigen A, the blood type is A If a person’s red blood cells have antigen B, the blood type is B. If the red blood cells have both antigen A and antigen B, the blood type is AB If the red blood cells have neither antigen A nor antigen B, the blood type is O. BY Mbuye Gilbert 16 Multiple alleles

17. The alleles for type A and type B blood are codominant; that is, both alleles are expressed. However , the allele for type O blood is recessive to both type A and type B Because a person has only two of the three alleles, the blood type varies depending on which two alleles are present For instance, if a person has the A allele and the B allele, the blood type is AB If a person has two A alleles, or one A and one O allele, the blood type is A If a person has two B alleles, or one B and one O allele, the blood type is B If a person has two O alleles, the blood type is O. BY Mbuye Gilbert 17 Multiple alleles

18. Although many characteristics are determined by alleles at a single place on the chromosome some characteristics are determined by an interaction of genes on several chromosomes or at several places on one chromosome This condition is polygenic inheritance. An example of polygenic inheritance is human skin color Genes for skin color are located in many places, and skin color is determined by which genes are present at these multiple locations A person with many genes for dark skin will have very dark skin color A person with multiple genes for light skin will have very light skin color Many people have some genes for light skin and some for dark skin, which explains why so many variations of skin color exist Height is another characteristic probably reflecting polygenic characteristics BY Mbuye Gilbert 18 Polygenic inheritance

19. Among the 23 pairs of chromosomes in human cells, one pair is the sex chromosomes. The remaining 22 pairs of chromosomes are referred to as autosomes The sex chromosomes determine the sex of humans Two types of sex chromosomes: the X chromosome and the Y chromosome . Females have two X chromosomes; males have one X and one Y chromosome. Typically , the female chromosome pattern is designated XX, while the male chromosome pattern is XY . Thus , the genotype of the human male would be 44 XY, while the genotype of the human female would be 44 XX (where 44 represents the autosomes). In humans, the Y chromosome is much shorter than the X chromosome Because of this shortened size, a number of sex-linked conditions occur BY Mbuye Gilbert 19 Sex linkage

20. When a gene occurs on an X chromosome, the other gene of the pair probably occurs on the other X chromosome Therefore, a female usually has two genes for a characteristic In contrast, when a gene occurs on an X chromosome in a male, there is usually no other gene present on the short Y chromosome Therefore, in the male, whatever gene is present on the X chromosome will be expressed BY Mbuye Gilbert 20 Sex linkage

21. A chromosome has many thousands of genes T here are an estimated 20,000 to 30,000 genes in the human genome A locus (plu Loci) is the physical site where a gene is located on a chromosome Inheritance involves the transfer of chromosomes from parent to offspring through meiosis and sexual reproduction It is common for a large number of genes to be inherited together if they are located on the same chromosome Genes that are inherited together are said to form a linkage group The concept of transfer of a linkage group is gene linkage. Gene linkage can show how close two or more genes are to one another on a chromosome The closer the genes are to each other, the higher the probability that they will be inherited together Crossing over occurs during meiosis, but genes that are close to each other tend to remain together during crossing over BY Mbuye Gilbert 21 Gene linkage

22. Chromosome mapping means determining the relative positions of gene in a chromosome by creating maps that are used to organize and understand genetic information on chromosomes These maps show the positions of genes and the distances between them based on a specific scale Chromosome mapping helps to understand the genome’s organization, structure, and function BY Mbuye Gilbert 22 Chromosome mapping

23. Genetic Mapping Genetic mapping, also known as linkage mapping Refers to the method used to create genetic maps to estimate the positions of genes in chromosomes These genetic maps are constructed by analyzing recombination patterns that occur during the crossing over of chromosomes.  Genetic mapping starts by using linkage analysis to study the frequency of recombination between genes.  Recombination frequency indicates genetic linkage and helps to determine whether the genes are linked together or not BY Mbuye Gilbert 23 Types of Chromosome Mapping

24. A centimorgan ( cM ) is a unit that describes a recombination frequency of 1%. As the distance between two genes increases, the likelihood of recombination occurring also increases, resulting in a higher recombination frequency between them. The recombination frequency is determined by using the number of recombinant individuals compared to the total number of offspring This frequency is expressed as a percentage BY Mbuye Gilbert 24 Genetic Mapping

25. Mendel’s law of independent assortment states that pairs of alleles segregate independently However, upon the rediscovery of Mendel’s work, it was known that some pairs of genes were inherited together due to their location on the same chromosome This phenomenon is known as genetic linkage  Linked genes are located close to each other on a chromosome, which increases the chance of them being inherited together So, the concept of linkage is used to understand the relative positions of genes on a chromosome. However, not all genes on a chromosome are necessarily linked Genes that are located further apart from each other have a higher chance of being separated during recombination BY Mbuye Gilbert 25 Genetic Linkage and Recombination

26. Recombination occurs during meiosis where homologous chromosomes pair up to form bivalents, and crossing-over can occur between chromatids , leading to the exchange of genetic material. Genes that are closer together have a lower chance of crossover Thus, the crossover percentage reflects the distance between the genes BY Mbuye Gilbert 26 Continuation

27. Restriction Fragment Length Polymorphisms (RFLPs)  are one of the earliest molecular markers used for genetic mapping RFLPs are specific patterns of DNA fragments created by restriction enzymes that identify and cut DNA at specific sites In genomic DNA, there can be variations in these restriction sites, leading to differences in the restriction patterns between closely related genomes RFLP can be mapped on a genome by studying its inheritance pattern.  Simple Sequence Length Polymorphisms (SSLPs)  are DNA markers that consist of repetitive sequences with variations in the number of repeats There are two types of SSLPs: minisatellites and microsatellites Minisatellites  are also known as the variable number of tandem repeats (VNTRs) and they have longer repeat units, up to 25 base pairs Microsatellites , also known as simple tandem repeats (STRs), have shorter repeat units, usually dinucleotide or tetranucleotide units. BY Mbuye Gilbert 27 Three commonly used molecular markers in genetic mapping

28. Single Nucleotide Polymorphisms (SNPs)  are variations in a genome with different nucleotides at specific positions SNPs can be detected using oligonucleotide hybridization, which allows selective pairing between an oligonucleotide and a DNA molecule. Techniques like DNA chip technology and solution hybridization have been developed for SNP screening, which offers faster and more efficient detection than gel-based methods. Limitations of Genetic Mapping Genetic mapping does not provide information about the physical distance between genes as it provides only the outline. Recombination levels can differ across various parts of the genome, some parts exhibit high rates of recombination, known as recombination hotspots, which can impact the accuracy of genetic mapping.  In organisms where obtaining a large number of progenies is difficult, the resolution of a genetic map is limited because it depends on the number of observed crossovers BY Mbuye Gilbert 28 Three commonly used molecular markers in genetic mapping

29.   Physical mapping involves determining the precise locations of DNA sequences on chromosomes It uses base pair as the unit of measurement.  Physical maps provide a direct representation of the physical structure of a chromosome and the positions of genes along its DNA sequence.  Markers used in physical mapping include ESTs (Expressed Sequence Tags), STS (Sequence Tagged Site) markers, and genome-wide DNA sequences. BY Mbuye Gilbert 29 Physical Mapping

30. Cytogenetic Mapping  is a method used to locate genes on chromosomes by using their unique banding patterns. Each chromosome has its own distinctive pattern of light and dark bands when observed under a microscope These bands are numbered to help in the precise identification of specific regions within a chromosome Cytogenetic maps can be created by correlating the position of a gene with the corresponding band on the chromosome These maps are also called idiograms .  Restriction Mapping  is a physical mapping method used to identify the positions of restriction sites on a DNA fragment Restriction mapping involves comparing the sizes of DNA fragments created by two different restriction enzymes Using RFLPs as markers for genetic mapping helps us to find the locations of certain variable restriction sites in a genome However, most restriction sites are not variable, so RFLPs cannot be used to map many sites. Restriction mapping can be used as an alternative method in order to locate non-variable restriction sites.  BY Mbuye Gilbert 30 Methods of Physical Mapping

31. Fluorescence in situ hybridization (FISH)  is a method used to visualize the position of specific DNA sequences on chromosomes. It involves labeling DNA probes and hybridizing them into intact chromosomes. In early versions of FISH, radioactive probes were used, but later fluorescent DNA labels were developed, which offer improved sensitivity and resolution.  Sequence Tagged Site (STS) Mapping  is a powerful mapping method for creating detailed physical maps of large genomes It involves using short DNA sequences called STSs, typically 100 to 500 base pairs long, that occur uniquely in the genome. The map can be created by identifying the fragments that contain specific STSs using hybridization analysis or PCR. STSs can be obtained from various sources, including expressed sequence tags (ESTs), SSLPs, or random genomic sequences obtained through sequencing cloned genomic DNA A commonly used marker for STS mapping is the EST, which is a unique sequence obtained by partially sequencing clones from a cDNA library.  BY Mbuye Gilbert 31 Methods of Physical Mapping

32. Radiation Hybrid (RH) Mapping  is another physical mapping method that estimates the distance between genetic markers. RH mapping uses radiation to break DNA fragments. By controlling the radiation exposure, this method can create breaks between linked markers, resulting in a more detailed map. RH mapping is valuable for locating various genetic markers and genomic fragments, particularly in regions with limited availability of highly polymorphic markers Limitations of Physical Mapping DNA fragments can be incorrectly mapped due to fragment breakage, deletion during replication, or contamination with host genetic material. There may be missing or incomplete coverage of DNA fragments in the mapping process, resulting in gaps. Restriction mapping method cannot be applied to large genomes.  Physical mapping using FISH is difficult to carry out and the process of accumulating data is slow. In a single experiment, only a limited number of map positions can be obtained. BY Mbuye Gilbert 32 Methods of Physical Mapping

33. Animal development depends on the differential expression of a constant genome to produce diverse cell types during embryogenesis. A typicalanimalgenomecontainsapproximately20,000genes. This is not only true for comparatively simple creatures such as nematode worms, but also pertains to the “crown and summit” of animal evolution, the human genome Differential gene expression can be defined as the synthesis of a protein (or RNA in the case of non-coding genes) in a subset of the cells comprising an embryo Differential expression most commonly hinges on de novo transcription. Thus, the b- globin gene is selectively expressed in developing red blood cells, but not other tissues, because the gene is transcribed only in blood cells However, there are examples of post-transcriptional mechanisms of differential gene expression BY Mbuye Gilbert 33 The role of genes in development

34. The most spectacular demonstration of“ genetic equivalence” among the different tissues of a developing animal is the transformation of virtually any cell type into an induced pluripotent stem ( iPS ) cell. Most mammalian embryos, including the human fetus, contain a small group of cells, the inner cell mass (ICM), which form all of the tissues and organs of the adult. The ICM cells are said to be “ pluripotent ” because they can produce many different cell types. The formation of ICM cells depends on the activities of three sequence-specific transcription factors—Oct4, Sox2, and Nanog The forced expression of these three factors in a differentiated cell type, such as a fibroblast cell (connective tissue), is sufficient to transform them into iPS cells, which have the properties of ICM cells BY Mbuye Gilbert 34 The role of genes in development

35. DNA biosynthesis proceeds in the 5'- to 3'-direction. This makes it impossible for DNA polymerases to synthesize both strands simultaneously. A portion of the double helix must first unwind, and this is mediated by  helicase  enzymes. The leading strand is synthesized continuously but the opposite strand is copied in short bursts of about 1000 bases, as the lagging strand template becomes available The resulting short strands are called  Okazaki fragments  (after their discoverers, Reiji and Tsuneko Okazaki) Bacteria have at least three distinct DNA polymerases: Pol I, Pol II and Pol III; it is Pol III that is largely involved in chain elongation. Strangely, DNA polymerases cannot initiate DNA synthesis  de novo , but require a short primer with a free 3'-hydroxyl group BY Mbuye Gilbert 35 DNA replication and transcription

36. This is produced in the lagging strand by an RNA polymerase (called DNA primase ) that is able to use the DNA template and synthesize a short piece of RNA around 20 bases in length. Pol III can then take over, but it eventually encounters one of the previously synthesized short RNA fragments in its path. At this point Pol I takes over, using its 5'- to 3'-exonuclease activity to digest the RNA and fill the gap with DNA until it reaches a continuous stretch of DNA. This leaves a gap between the 3'-end of the newly synthesized DNA and the 5'-end of the DNA previously synthesized by Pol III. The gap is filled by DNA ligase , an enzyme that makes a covalent bond between a 5'-phosphate and a 3'-hydroxyl group The initiation of DNA replication at the leading strand is more complex and is discussed in detail in more specialized texts. BY Mbuye Gilbert 36 DNA replication and transcription

37. BY Mbuye Gilbert 37 DNA replication

38. BY Mbuye Gilbert 38 DNA replication

39. DNA replication is not perfect. Errors occur in DNA replication, when the incorrect base is incorporated into the growing DNA strand. This leads to  mismatched  base pairs, or  mispairs . DNA polymerases have proofreading activity, and a  DNA repair  enzymes have evolved to correct these mistakes. Occasionally, mispairs survive and are incorporated into the genome in the next round of replication. These mutations may have no consequence, they may result in the death of the organism They may result in a genetic disease or cancer; or they may give the organism a competitive advantage over its neighbours , which leads to evolution by natural selection. BY Mbuye Gilbert 39 Mistakes in DNA replication

40. Transcription is the process by which DNA is copied ( transcribed ) to mRNA, which carries the information needed for protein synthesis. Transcription takes place in two broad steps. First, pre-messenger RNA is formed, with the involvement of RNA polymerase enzymes. The process relies on Watson-Crick base pairing, and the resultant single strand of RNA is the reverse-complement of the original DNA sequence. The pre-messenger RNA is then "edited" to produce the desired mRNA molecule in a process called  RNA splicing . BY Mbuye Gilbert 40 Transcription

41. The mechanism of transcription has parallels in that of  DNA replication As with DNA replication, partial unwinding of the double helix must occur before transcription can take place, and it is the RNA polymerase enzymes that catalyze this process. Unlike DNA replication, in which both strands are copied, only one strand is transcribed. The strand that contains the gene is called the  sense  strand, while the complementary strand is the  antisense  strand. The mRNA produced in transcription is a copy of the sense strand, but it is the antisense strand that is transcribed. Ribonucleoside triphosphates (NTPs) align along the antisense DNA strand, with Watson-Crick base pairing (A pairs with U). RNA polymerase joins the ribonucleotides together to form a pre-messenger RNA molecule that is complementary to a region of the antisense DNA strand Transcription ends when the RNA polymerase enzyme reaches a triplet of bases that is read as a "stop" signal. The DNA molecule re-winds to re-form the double helix. BY Mbuye Gilbert 41 Formation of pre-messenger RNA

42. BY Mbuye Gilbert 42 Formation of pre-messenger RNA

43. The pre-messenger RNA thus formed contains introns which are not required for protein synthesis. The pre-messenger RNA is chopped up to remove the introns and create messenger RNA (mRNA) in a process called RNA splicing BY Mbuye Gilbert 43 RNA splicing

44. In reverse transcription, RNA is "reverse transcribed" into DNA. This process, catalyzed by reverse transcriptase enzymes, allows retroviruses, including the human immunodeficiency virus (HIV), to use RNA as their genetic material. Reverse transcriptase enzymes have also found applications in biotechnology, allowing scientists to convert RNA to DNA for techniques such as  PCR BY Mbuye Gilbert 44 Reverse transcription

45. The genetic code can be defined as the set of certain rules using which the living cells translate the information encoded within genetic material (DNA or mRNA sequences) In other words, genetic code is defined as the nucleotide sequence of the base on DNA which is translated into a sequence of amino acids of the protein to be synthesized The  ribosomes   are responsible to accomplish the process of translation. They link the amino acids in an mRNA-specified (messenger RNA) order using tRNA (transfer RNA ) molecules to carry amino acids and to read the mRNA three nucleotides at a time A  codon  is a sequence of three nucleotides which together form a unit of genetic code in a DNA or RNA molecule. BY Mbuye Gilbert 45 The Genetic code

46. The Genetic code The genetic code is almost universal It is the basis of the transmission of hereditary information by nucleic acids in all organisms There are four bases in RNA (A,G,C and U) so there are 64 possible triplet codes (4 3  = 64). In theory only 22 codes are required: one for each of the 20 naturally occurring amino acids, with the addition of a start codon and a stop codon (to indicate the beginning and end of a protein sequence). Many amino acids have several codes ( degeneracy ), so that all 64 possible triplet codes are used. For example Arg and Ser each have 6 codons whereas Trp and Met have only one. No two amino acids have the same code but amino acids whose side-chains have similar physical or chemical properties tend to have similar codon sequences, e.g. the side-chains of Phe , Leu , Ile, Val are all hydrophobic, and Asp and Glu are both carboxylic acids BY Mbuye Gilbert 46 The genetic code, mutation, mutagenesis and repair

47. This means that if the incorrect tRNA is selected during translation (owing to mispairing of a single base at the codon-anticodon interface) the misincorporated amino acid will probably have similar properties to the intended tRNA molecule. Although the resultant protein will have one incorrect amino acid it stands a high probability of being functional. Organisms show " codon bias" and use certain codons for a particular amino acid more than others. For example, the codon usage in humans is different from that in bacteria; it can sometimes be difficult to express a human protein in bacteria because the relevant tRNA might be present at too low a concentration. BY Mbuye Gilbert 47 The Genetic code

48. Triplet code Non-ambiguous and Universal Degenerate code Nonoverlapping code Commaless Start and Stop Codons Polarity BY Mbuye Gilbert 48 Properties of Genetic Code The complete set of relationships among  amino acids  and  codons   is said to be a genetic code which is often summarized in a table.  

49. In the living cell, DNA undergoes frequent chemical change, especially when it is being replicated (in S phase of the eukaryotic cell cycle). Most of these changes are quickly repaired. Those that are not result in a mutation. Thus, mutation is a failure of DNA repair BY Mbuye Gilbert 49 Mutation Hemoglobin sequence

50. Single-base substitutions A single base, say an A, becomes replaced by another Single base substitutions are also called point mutations. If one purine [A or G] or pyrimidine [C or T] is replaced by the other, the substitution is called a transition . If a purine is replaced by a pyrimidine or viceversa , the substitution is called a tra nsversion ) Missense mutations With a missense mutation, the new nucleotide alters the codon so as to produce an altered amino acid in the protein product. EXAMPLE: sickle-cell disease; The replacement of A by T at the 17th nucleotide of the gene for the beta chain of hemoglobin changes the codon GAG (for glutamic acid) to GTG (which encodes valine ). Thus the 6th amino acid in the chain becomes valine instead of glutamic acid. Another example: Patient A with cystic fibrosis. BY Mbuye Gilbert 50 Types of Mutations

51. Nonsense mutations With a nonsense mutation, the new nucleotide changes a codon that specified an amino acid to one of the STOP codons (TAA, TAG, or TGA) Therefore, translation of the messenger RNA transcribed from this mutant gene will stop prematurely. The earlier in the gene that this occurs, the more truncated the protein product and the more likely that it will be unable to function Silent mutations Most amino acids are encoded by several different codons For example, if the third base in the TCT codon for serine is changed to any one of the other three bases, serine will still be encoded Such mutations are said to be silent because they cause no change in their product and cannot be detected without sequencing the gene (or its mRNA). BY Mbuye Gilbert 51 Types of Mutations

52. Splice-site mutations The removal of intron sequences, as pre-mRNA is being processed to form mRNA, must be done with great precision. Nucleotide signals at the splice sites guide the enzymatic machinery If a mutation alters one of these signals, then the intron is not removed and remains as part of the final RNA molecule. The translation of its sequence alters the sequence of the protein product BY Mbuye Gilbert 52 Continuation

53. Insertions and Deletions (Indels) Extra base pairs may be added (insertions) or removed (deletions) from the DNA of a gene. The number can range from one to thousands Collectively, these mutations are called indels Indels involving one or two base pairs (or multiples thereof) can have devastating consequences to the gene because translation of the gene is " frameshifted “ The mRNA is translated in new groups of three nucleotides and the protein specified by these new codons will be worthless Frameshifts often create new stop codons and thus generate nonsense mutations BY Mbuye Gilbert 53

54. Insertions and Deletions (Indels) Frameshifts often create new stop codons and thus generate nonsense mutations Indels of three nucleotides or multiples of three may be less serious because they preserve the reading frame However, a number of inherited human disorders are caused by the insertion of many copies of the same triplet of nucleotides. Huntington's disease and the fragile X syndrome are examples of such trinucleotide repeat diseases Other types of mutations include; Duplication, translocations, etc BY Mbuye Gilbert 54

55. BY Mbuye Gilbert 55

56. Many agents (physical, chemical and environmental) have the mutagenic properties to cause mutations. They are known as mutagens. Mutagenic agents induce mutation in either of the following ways They may replace in the DNA. They may alter the base in such a way that it specifically mispairs with another base. They may damage the base so much that it can no longer pair with any base. They may intercalate themselves in the DNA paving way for addition or deletion of bases. BY Mbuye Gilbert 56 MUTAGENESIS

57. These consist of high energy radiations which could penetrate living cells and affect the genetic material The effect of radiations on living cells and tissues is directly proportional to the degree of penetration of the radiation Radiations are of two types viz. electro-magnetic radiations and particulate radiations X-rays, gama -rays and UV rays are short wavelength electromagnetic radiations which penetrate cells and tissues strongly. Penetration power of electromagnetic radiation is inversely propotional to its wave length. Particulate radiations are in the form of subatomic particles emitted from atoms with high energy. Alpha particles, beta particles and neutrons fall in this category Alpha particles and beta-particles are charged particles However, beta particles being smaller in size are more penetrating than alpha particles Neutrons ejected from radioactive isotopes do not carry any charge and hence are not deflected when they travel through living matter. Thus they are extremely penetrant and can cause severe damage to the living tissues as well as genetic material. BY Mbuye Gilbert 57 Physical Mutagens

58. The physical mutagens are also divided as high energy ionizing radiations which include cosmic rays, X-rays, gamma-rays and particulate radiations and low energy non-ionizing radiations which include ultraviolet light. The high energy radiations create ionization in the living cells. While passing through cells and tissues they collide with molecules such as water and cause the expulsion of electrons This expulsion creates a positively charged ions The ejected electron can not remain in the free state and therefore, is picked up by another ion creating a negative ion The generation of free ions by radiations is the basis of extensive damage caused by them at the somatic and gametic level These ions may combine with oxygen producing highly reactive chemical which may act on genes, chromosomes and other parts of the cells. Peroxides which are mutagenic may be formed in the presence of oxygen following the splitting of water BY Mbuye Gilbert 58 Physical Mutagens

59. Experiments have shown that sensitivity to a radiation and the rate of mutation are much lower in the organisms maintained in oxygen-free environment. Non-ionizing radiations such as UV rays have major effect in the formation of dimers whereby adjacent pyrimidine bases become linked to one another by carbon to carbon bonds Dimerization results in intra strand or inter strand cross linking which distorts the DNA conformation, thereby affecting the normal replication. Many of these radiations are used in medicine, agriculture and warfare. So, working out dose effect relationship becomes important Experiments have suggested, there is no dose which may be absolutely ineffective Each doubling of dose results into the doubling of mutations induced BY Mbuye Gilbert 59 Physical Mutagens

60. Base Analogues Base analogues are the chemicals that have molecular structure that are extremely similar to bases of DNA These chemicals act as mutagens and during DNA replication get incorporated so as to form base pairs with usual bases One such chemical is 5- bromouracil (5 BU) 5 BU is a base analogue of thymine and usually pairs with adenine The bromine atom in 5 BUdR so alters the charge distribution of the molecule that it may tautomerise to a 5 BUdR * form quite frequently After tautomerisation it possesses the base pairing properties of cytosine, that is, it behaves like cytosine. You can see in Fig. 9.7 that the shift generates G A → transition. When a base pair change causes the change in mRNA codon resulting into a modified protein in place of one specified by wild-type, a missense mutation occurs BY Mbuye Gilbert 60 Chemical Mutagens

61. Nonsense mutation occurs when base pair changes result in the change in mRNA that generate a chain terminating codon which leads to premature termination of protein synthesis Neutral mutation may go unnoticed as it is a base pair change that changes a codon in mRNA such that the resulting amino acid substitution does not a alter the function of the protein For example, change from codon AGG to AAG which substitutes aminoacid lysine for arginine . Both amino acids are similar in properties, so function of protein is not altered significantly. When base pair change alters a codon in mRNA which may still code for same amino acid, silent mutation occurs For example, change from mRNA codon AGG to AGA both of which specify arginine BY Mbuye Gilbert 61 Base Analogues

62. Many chemicals [e.g. nitrous acid, hydroxylamine, ethyl methane sulfonate (EMS) are known to change the base sequence in DNA Nitrous acid (-HNO2) and hydroxylamine replace amino group (-NH2) by hydroxyl group (-OH) which leads to deamination of nitrogenous bases For example, deamination of cytosine produces uracil In case, uracil is not repaired back, it will direct the incorporation of adenine in the new DNA strand during replication This ultimately results in conversion of CG base pair to a TA base pair i.e. a Transition mutation. BY Mbuye Gilbert 62 Deamination and Depurination

63. Depurination Transversions as discussed earlier in this unit are caused by certain alkylating agents (e.g. ethyl methane sulfonate ; EMS). The removal of purine from the strand of DNA is called depurination During DNA replication, any base can get inserted in the complementary strand if depurination occurs Next cycle of DNA replication leads to trans version This leads to genetic mutation which is irreversible BY Mbuye Gilbert 63 5-Bromouracil and its mutagenic effect

64. Alkylating Agents : Such as ethyl methane sulfonate (EMS), ethyl ethane sulfonate (EES) and diethyl sulphate (DES) act on DNA by adding alkyl group (ethyl or methyl) to all four bases. However, these agents show a strong preference for base guanine. This results either in mispairing of affected base or its loss entirely, creating a gap thus causing mutations. Intercalating Agents: This type of mutagen includes ethidium bromide and Acridine dyes ( proflavin and acridine orange). Intercalating agents can mimic base pairs and slip between the base pairs in double helix and open the helix which leads to increase in distance between base pairs This results in deletion or addition of base pairs during DNA replication. BY Mbuye Gilbert 64 Chemical Mutagens

65. BY Mbuye Gilbert 65 Some common mutagens and their properties

66. Damaged or inappropriate bases can be repaired by several mechanisms: Direct chemical reversal of the damage Excision Repair, in which the damaged base or bases are removed and then replaced with the correct ones in a localized burst of DNA synthesis There are three modes of excision repair, each of which employs specialized sets of enzymes. 1 . Base Excision Repair (BER) 2. Nucleotide Excision Repair (NER) 3. Mismatch Repair (MMR) BY Mbuye Gilbert 66 DNA repair

67. Direct Reversal of Base Damage Perhaps the most frequent cause of point mutations in humans is the spontaneous addition of a methyl group (CH3-) (an example of alkylation) to Cs followed by deamination to a T Fortunately, most of these changes are repaired by enzymes, called glycosylases , that remove the mismatched T restoring the correct C This is done without the need to break the DNA backbone (in contrast to the mechanisms of excision repair described below) Some of the drugs used in cancer chemotherapy ("chemo") also damage DNA by alkylation Some of the methyl groups can be removed by a protein encoded by our MGMT gene However, the protein can only do it once, so the removal of each methyl group requires another molecule of protein. BY Mbuye Gilbert 67 DNA repair

68. This illustrates a problem with direct reversal mechanisms of DNA repair: they are quite wasteful. Each of the myriad types of chemical alterations to bases requires its own mechanism to correct. What the cell needs are more general mechanisms capable of correcting all sorts of chemical damage with a limited toolbox This requirement is met by the mechanisms of excision repair BY Mbuye Gilbert 68 Direct Reversal of Base Damage

69. 1. Removal of the damaged base (estimated to occur some 20,000 times a day in each cell in our body!) by a DNA glycosylase . We have at least 8 genes encoding different DNA glycosylases each enzyme responsible for identifying and removing a specific kind of base damage. 2. removal of its deoxyribose phosphate in the backbone, producing a gap. We have two genes encoding enzymes with this function. 3. replacement with the correct nucleotide. This relies on DNA polymerase beta, one of at least 11 DNA polymerases encoded by our genes. 4. ligation of the break in the strand Two enzymes are known that can do this; both require ATP to provide the needed energy BY Mbuye Gilbert 69 Base Excision Repair (BER)

70. NER differs from BER in several ways It uses different enzymes Even though there may be only a single "bad" base to correct Its nucleotide is removed along with many other adjacent nucleotides; that is, NER removes a large "patch" around the damage The steps and some key players: The damage is recognized by one or more protein factors that assemble at the location. The DNA is unwound producing a "bubble". The enzyme system that does this is Transcription Factor IIH, TFIIH, (which also functions in normal transcription). BY Mbuye Gilbert 70 Nucleotide Excision Repair (NER)

71. Cuts are made on both the 3' side and the 5' side of the damaged area so the tract containing the damage can be removed. A fresh burst of DNA synthesis — using the intact (opposite) strand as a template — fills in the correct nucleotides. The DNA polymerases responsible are designated polymerase delta and epsilon. A DNA ligase covalent binds the fresh piece into the backbone. BY Mbuye Gilbert 71

72. Mismatch repair deals with correcting mismatches of the normal bases; that is, failures to maintain normal Watson-Crick base pairing (A•T, C•G) It can enlist the aid of enzymes involved in both baseexcision repair (BER) and nucleotide-excision repair (NER) as well as using enzymes specialized for this function. Recognition of a mismatch requires several different proteins including one encoded by MSH2 Cutting the mismatch out also requires several proteins, including one encoded by MLH1 Mutations in either of these genes predispose the person to an inherited form of colon cancer So these genes qualify as tumor suppressor genes. BY Mbuye Gilbert 72 Mismatch Repair (MMR)

73. Synthesis of the repair patch is done by the same enzymes used in NER: DNA polymerase delta and epsilon Cells also use the MMR system to enhance the fidelity of recombination; i.e., assure that only homologous regions of two DNA molecules pair up to crossover and recombine segments (e.g., in meiosis). BY Mbuye Gilbert 73 Mismatch Repair (MMR)

74. Ionizing radiation and certain chemicals can produce both single-strand breaks (SSBs) and double-strand breaks (DSBs) in the DNA backbone. Single-Strand Breaks (SSBs): Breaks in a single strand of the DNA molecule are repaired using the same enzyme systems that are used in Base-Excision Repair (BER). Double-Strand Breaks (DSBs): There are two mechanisms by which the cell attempts to repair a complete break in a DNA molecule Direct joining of the broken ends. This requires proteins that recognize and bind to the exposed ends and bring them together for ligating . They would prefer to see some complementary nucleotides but can proceed without them so this type of joining is also called Nonhomologous End-Joining (NHEJ) Errors in direct joining may be a cause of the various translocations that are associated with cancers Examples: o Burkitt's lymphoma o the Philadelphia chromosome in chronic myelogenous leukemia (CML) o B-cell leukemia BY Mbuye Gilbert 74 Repairing Strand Breaks

75. It is the process of synthesis of proteins from messenger RNA transcripts (mRNA) after the process of transcription of DNA to RNA. It takes place in the cytoplasm by specialized organelle known as ribosomes .  There are no endoplasmic reticulum in the prokaryotes and ribosomes are suspended in the cytoplasm, whereas endoplasmic reticulum are present in eukaryotes which harbors ribosomes Translation takes place on rough endoplasmic reticulum (RER) in eukaryotes, whereas translation occurs freely in cytoplasm in the prokaryotes The codons on the mRNA are translated into amino acid sequence which leads to the synthesis of protein. Translation requires a variety of cellular components, such as proteins, RNAs and different small molecules. It has also three main steps: BY Mbuye Gilbert 75 Protein synthesis (bacteria and Eukaryotes)

76. o Initiation – Formation of mRNA-ribosome complex o Elongation – Formation of polypeptide chain complimentary to the mRNA o Termination – Termination of polypeptide chain The “Central Dogma” It states that genetic information is transmitted form DNA to RNA to protein and this information cannot be transferred back from protein to either protein or nucleic acid”. BY Mbuye Gilbert 76

77. BY Mbuye Gilbert 77 Proteins are the “workhorse” molecule found in organisms, which determine phenotype of an organism, i.e. what we look like. Proteins are made of polypeptide chains that have primary, secondary, tertiary and quaternary structure. The polypeptide chains are made of 20 amino acids - an average polypeptide chain is 400 amino acid long (can be shorter than this value). The part of the DNA that codes for a particular polypeptide chain is called as a gene

78. BY Mbuye Gilbert 78 Overview of Protein Synthesis DNA is transcribed to mRNA in the cytoplasm in prokaryotes as it has no nucleus, and in the nucleus in eukaryotes. With mRNA and help of other RNA as well as protein molecules, mRNA is translated into specific proteins in the cytoplasm Therefore, RNA is the intermediate between the DNA code and the actual synthesis of a protein

79. BY Mbuye Gilbert 79 RNAs in the Protein Synthesis

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81. BY Mbuye Gilbert 81 Protein Synthesis in Prokaryotes and Eukaryotes

82. BY Mbuye Gilbert 82 How are the codons matched to amino acid?

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88. Although the genetic code has codons for only 20 amino acids, many other amino acids are occasionally found in proteins. Apart from selenocysteine and pyrrolysine these extra amino acids are made by modifying genetically encoded amino acids after the polypeptide chain has been assembled. This is known as post-translational modification An example of medical importance is diphthamide , which is derived from histidine by post-translational modification It is found only in elongation factor eEF2 of eukaryotes and Archaea , in a region of the amino acid sequence that is highly conserved. The corresponding bacterial factor, EF-G, does not contain diphthamide . Diphthamide was named after diphtheria, an infectious disease caused by the bacterium Corynebacterium diphtheriae Diphtheria toxin attaches an ADP-ribose fragment to elongation factor eEF2 via diphthamide and this inhibits protein synthesis and kills the target cells eEF2 normally splits GTP and uses the energy released to move the peptidyl -tRNA from the A-site to the P-site. ADP- ribosylated eEF2 still binds GTP but cannot hydrolyze it or translocate the peptidyl -tRNA. BY Mbuye Gilbert 88 Post-Translational Modifications of Proteins

89. BY Mbuye Gilbert 89 Post-Translational Modifications of Proteins

90. Gene cloning The production of exact copies of a particular gene or  DNA  sequence using genetic engineering techniques is called gene cloning. The term “gene cloning,” “DNA cloning,” “molecular cloning,” and “recombinant DNA technology” all refer to same technique. When DNA is extracted from an organism, all its genes are obtained In gene (DNA) cloning a particular gene is copied forming “clones”. Cloning is one method used for isolation and amplification of gene of interest DNA cloning can be achieved by two different methods: Cell based DNA cloning Cell-free DNA cloning (PCR) BY Mbuye Gilbert 90 Gene cloning andrestriction endonucleases

91. DNA fragment  containing the desired genes to be cloned. Restriction enzymes  and  ligase enzymes . Vectors  – to carry, maintain and replicate cloned gene in host cell. Host cell – in which recombinant DNA can replicate. BY Mbuye Gilbert 91 Requirements for Gene Cloning (Cell-based)

92. A fragment of DNA, containing the gene to be cloned, is inserted into a suitable vector, to produce a recombinant DNA molecule . The vector acts as a vehicle that transports the gene into a host cell usually a bacterium, although other types of living cell can be used. Within the host cell the vector multiplies, producing numerous identical copies not only of itself but also of the gene that it carries. When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place. After a large number of cell divisions, a colony, or clone, of identical host cells is produced Each cell in the clone contains one or more copies of the recombinant DNA molecule; the gene carried by the recombinant molecule is now said to be cloned. BY Mbuye Gilbert 92 Principle of Gene Cloning

93. The basic 7 steps involved in gene cloning are: Isolation of DNA [gene of interest] fragments to be cloned. Insertion of isolated DNA into a suitable vector to form recombinant DNA Introduction of recombinant DNA into a suitable organism known as host. Selection of transformed host cells and identification of the clone containing the gene of interest Multiplication/Expression of the introduced Gene in the host. Isolation of multiple gene copies/Protein expressed by the gene. Purification of the isolated gene copy/protein BY Mbuye Gilbert 93 Steps in Gene Cloning

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95. The target DNA or gene to be cloned must be first isolated A gene of interest is a fragment of gene whose prod­uct (a protein, enzyme or a hormone) interests us . For example, gene encoding for the hormone insulin. The desired gene may be isolated by using restriction endonuclease (RE) enzyme, which cut DNA at specific recognition nucleotide se­quences known as restriction sites towards the inner region (hence endonuclease) producing blunt or sticky ends. Sometimes, reverse transcriptase enzyme may also be used which synthesizes complementary DNA strand of the desired gene using its mRNA BY Mbuye Gilbert 95 A . Isolation of the DNA fragment or gene

96. The vector is a carrier molecule which can carry the gene of interest (GI) into a host, replicate there along with the GI making its multiple copies. The cloning vectors are limited to the size of insert that they can carry Depending on the size and the application of the insert the suitable vector is selected. The different types of vectors available for cloning  are plasmids, bacteriophages , bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs)  and  mammalian artificial chromosomes (MACs). However, the most commonly used cloning vectors include plasmids and bacteriophages (phage λ) beside all the other available vectors BY Mbuye Gilbert 96 B . Selection of suitable cloning vector

97. All cloning vectors are carrier DNA molecules . These carrier molecules should have few common features in general such as: It must be self-replicating inside host cell. It must possess a unique restriction site for RE enzymes. Introduction of donor DNA fragment must not interfere with replication property of the vector. It must possess some marker gene such that it can be used for later identification of recombinant cell (usually an antibiotic resistance gene that is absent in the host cell). They should be easily isolated from host cell BY Mbuye Gilbert 97 C . Essential Characteristics of Cloning Vectors

98. The plasmid vector is cut open by the same RE enzyme used for isolation of donor DNA fragment. The mixture of donor DNA fragment and plasmid vector are mixed together. In the presence of DNA ligase , base pairing of donor DNA fragment and plasmid vector occurs. The result­ing DNA molecule is a hybrid of two DNA molecules – the GI and the vector. In the ter­minology of genetics this intermixing of dif­ferent DNA strands is called recombination. Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and the technology is referred to as the  recom­binant DNA technology . BY Mbuye Gilbert 98 D . Formation of Recombinant DNA

99. The recombinant vector is transformed into suitable host cell mostly, a bacterial cell. This is done either for one or both of the following reasons: To replicate the recombinant DNA mol­ecule in order to get the multiple copies of the GI. To allow the expression of the GI such that it produces its needed protein product. Some bacteria are naturally transformable; they take up the recombinant vector automatically. For example:  Bacillus ,  Haemophillus ,  Helicobacter pylori , which are naturally competent. Some other bacteria, on the other hand require the incorporation by artificial methods such as Ca ++  ion treatment, electroporation , etc. BY Mbuye Gilbert 99 E . Transformation of recombinant vector into suitable host

100. The transformation process generates a mixed population of transformed and non-trans- formed host cells. The selection process involves filtering the transformed host cells only. For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector is employed. For examples, PBR322 plasmid vector contains different marker gene ( Ampicillin resistant gene and Tetracycline resistant gene. When pst1 RE is used it knock out Ampicillin resistant gene from the plasmid, so that the recombinant cell become sensitive to Ampicillin . BY Mbuye Gilbert 100 F . Isolation of Recombinant Cells

101. Once transformed host cells are separated by the screening process; becomes necessary to provide them optimum parameters to grow and multiply. In this step the transformed host cells are introduced into fresh culture media . At this stage the host cells divide and re-divide along with the replication of the recom­binant DNA carried by them. If the aim is obtaining numerous copies of GI, then simply replication of the host cell is allowed But for obtaining the product of interest, favourable conditions must be provided such that the GI in the vector expresses the product of interest. BY Mbuye Gilbert 101 G . Multiplication of Selected Host Cells

102. BY Mbuye Gilbert 102 H . Isolation and Purification of the Product The next step involves isolation of the multiplied GI attached with the vector or of the protein encoded by it. This is followed by purification of the isolated gene copy/protein. Applications of Gene Cloning A particular gene can be isolated and its nucleotide sequence determined Control sequences of DNA can be identified & analyzed Protein/enzyme/RNA function can be investigated Mutations can be identified, e.g. gene defects related to specific diseases Organisms can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.

103. Restriction enzyme, also called restriction endonuclease, is a protein produced by bacteria that cleaves  DNA at specific sites along the molecule. Restriction endonucleases cut the DNA double helix in very precise ways . It cleaves DNA into fragments at or near specific recognition sites within the molecule known as restriction sites. They have the capacity to recognize specific base sequences on DNA and then to cut each strand at a given place. Hence , they are also called as ‘molecular scissors ’ BY Mbuye Gilbert 103 Restriction endonucleases

104. BY Mbuye Gilbert 104 Source of Restriction Enzymes The natural source of restriction endonucleases are bacterial cells. These enzymes are called restriction enzymes because they restrict infection of bacteria by certain viruses (i.e., bacteriophages ), by degrading the viral DNA without affecting the bacterial DNA. Thus , their function in the bacterial cell is to destroy foreign DNA that might enter the cell. The restriction enzyme recognizes the foreign DNA and cuts it at several sites along the molecule. Each bacterium has its own unique restriction enzymes and each enzyme recognizes only one type of sequence

105. BY Mbuye Gilbert 105 Recognition Sites

106. When a restriction endonuclease recognizes a particular sequence, it snips through the DNA molecule by catalyzing the hydrolysis (splitting of a chemical bond by addition of a water molecule) of the bond between adjacent nucleotides. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix Types of Restriction Enzymes Traditionally, four types of restriction enzymes are recognized, designated I, II, III, and IV, which differ primarily in structure, cleavage site, specificity, and cofactors.  Type I enzymes  cleave at sites remote from a recognition site; require both ATP and S- adenosyl -L- methionine to function; multifunctional protein with both restriction and methylase activities. Type II enzymes  cleave within or at short specific distances from a recognition site; most require magnesium; single function (restriction) enzymes independent of methylase . Type III enzymes  cleave at sites a short distance from a recognition site; require ATP (but do not hydrolyze it); S- adenosyl -L- methionine stimulates the reaction but is not required; it exists as part of a complex with a modification methylase . Type IV enzymes  target modified DNA, e.g. methylated , hydroxymethylated and glucosyl-hydroxymethylated DNA. BY Mbuye Gilbert 106 Mechanism of Cleavage of Restriction Enzymes

107. Since their discovery in the 1970s, many restriction enzymes have been identified while  Type II restriction enzymes have been characterized. Each enzyme is named after the bacterium from which it was isolated, using a naming system based on bacterial genus, species and strain. For example, the name of the  EcoRI  restriction enzyme was derived as: E –  Escherichia : Genus co-  coli:  specific species R-  RY13: strain I-  First identified: order of identification in the bacterium BY Mbuye Gilbert 107 Nomenclature of Restriction Enzymes

108. Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason, they are indispensable tools of recombinant DNA technology (genetic engineering). The most useful aspect of restriction enzymes is that each enzyme recognizes the same unique base sequence regardless of the source of the DNA. It means that these enzymes establish fixed landmarks along an otherwise very regular DNA molecule. This allows dividing a long DNA molecule into fragments that can be separated from each other by size with the technique of gel electrophoresis. Each fragment, thus generated, are also available for further analysis, including the sequencing. One value of cutting DNA molecule up into discrete fragments is being able to locate a particular gene on the fragment where it resides which is done by the general technique of Southern blotting. BY Mbuye Gilbert 108 Applications of Restriction Enzymes

109. One of the most popular restriction enzymes is called EcoRI from  E. coli  (bacterium). Hundreds of other restriction enzymes with different sequence specificities have been isolated from several bacteria and are commercially available BY Mbuye Gilbert 109 Some examples of Restriction Enzymes

110. BY Mbuye Gilbert 110 Cloning vectors and gene expression system

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