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Biology 110.6 Genetics (Chapter 9, 10, 11, 12)

1 - Introduction 2 - Mendel’s Principles 3 - The Chromosomal Basis of Inheritance 4 - The Genetic Material 5 - DNA Replication 6 - The Flow of Genetic Information 7 - The Control of Gene Expression 8 - DNA Technology 9 - The Genome Era 1 - Introduction Genetics - is the branch of biology concerned with heredity and variation of inherited characteristics (the transmission of genetic information from parent to offspring). Historical Account: The Greek influence: Hippocrates and Aristotle Hippocrates (the father of medicine; 500-400 B.C.) – Pangenesis – particles called pangenes travel from each part of an organism’s body to the eggs/sperm and are then passed to the offspring. Aristotle (the philosopher and naturalist, 384-322 B.C.) - What is inherited was formed from blood, rather than particles of the features themselves. The “vital heat” from blood had the capacity to produce offspring of the same form, not because it already contained the all parts in miniature. The dawn of modern biology: 1600-1850 The theory of epigenesis (English anatomist William Harvey, 1578-1657) - an organism is derived from substances present in the egg, which differentiate into adult structures during embryonic development. The atomic theory (John Dalton in 1808) – all matter is composed of small invisible units called atoms. The germ(cell) theory (Louis Pasteur, 1822-1895 etc) – all organisms are composed of basic visible units called cells, which are derived from similar preexisting structures. The fixity of species (Carolus Linnaeus, 1707-1778 etc) – the ideal that members of a species can give rise only to other members of the species, thus implying that all species are independently created.

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The theory of the origin of species and natural selection (Charles Darwin, 18091882) - species arose by descent with modification from other ancestral species. - the differential contribution of offspring to the next generation by various genetic types belonging to the same species. • •

Inheritance of acquired characters: it was believed that an organism acquires some traits during its life time and they are then passed on to the offspring. “Blending” theory of inheritance: the genetic materials contributed by the male and female parents mix in forming the offspring.

The modern science of genetics The principles (basic roles) of inheritance were first demonstrated by Gregor Mendel (1822-1884), an Austrian monk who bred pea plants. Mendel was raised on a farm. He worked on breeding of vegetables and fruits. After entering the monastery, he continued his study on mathematics and botany among others at the University of Vienna. Shortly after his university training, Mendel began his experiments with garden pea. He planned his experiments carefully, recorded the data, and subjected the results to mathematical analysis. Mendel was the first scientist to effectively apply quantitative methods to the study of inheritance. His major discoveries, including those now known as Mendel’s principles of segregation and independent assortment, became the foundation of the science of genetics. Although his work was unappreciated in his lifetime, it was rediscovered in 1900.

2 - Mendel’s Principles Mendel’s experimental materials The plant - garden pea (Pisium sativum). Mendel chose the materials for three reasons: • Pea plant has several varieties with clear distinguishable traits. • Pea plant can be cross pollinated by hands between varieties, it self alone would self pollinate. Thus, both cross- and self- pollinations are possible. • Pea plant could be easily cultivated in the garden. Single Trait Crosses: Mendel first performed experiments to determine the pattern of inheritance of single traits. Such experiments are called Monohybrid Cross.

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Mendel chose seven traits including flower colour, flower position, seed shape, seed colour, pod shape, pod colour, and plant height. • The traits chosen had pairs. • Pairs were clearly distinguished. • Varieties with these traits are true-breeding lines (a genetically pure line of organism; organisms for which sexual reproduction produces offspring with inherited trait(s) identical to those of parents). Cross/hybridization - the cross-fertilization of two different varieties/species. Hybrid - the offspring of parents (P generation) of two different varieties/species. F1 generation - the first generation (hybrid) resulting from a cross (F1 stands for first filial). F2 generation - the next generation of plants from self-fertilization of the F1 offspring. Experiments & Results (i.e. flower colour) F1 generation: When he crossed a plant with purple flowers to a plant with white flowers, regardless of which of the parents provided, all progeny produced plants with purple flowers. Notes: 1. No blending of traits observed. 2. What happened to the white flower trait? Lost or not allowed to be expressed? F2 generation: • Produced both purple and white flower plants. • Out of 929 plants, 705 purple flowers and 224 white flowers, a ratio of 3:1 Mendel tested all seven traits. In all cases, in F1 generation only one of the two traits was expressed, and in F2, 75% of the generation was the same as in F1, and 25% the other. Mendel proposed: i. For each trait there is a pair (alleles) of heritable factors (genes) in each parent. ii. During the formation of gametes(♀ or ♂ sex cells), the two factors for a trait separate, and only one factor is passed on to a gamete. iii. When ♀ or ♂ gametes fuse during fertilization, each parent contributes one of the factors for a trait, so that the offspring has a pair of factors. iv. For a cross between individuals with different traits of a pair, factors for both traits are present but one is masked by its partner. Alleles - alternative forms of a gene; genes governing variation of the same character that occupy corresponding positions (loci) on homologous chromosomes. Dominant allele - an allele that is always expressed when it is present, regardless of whether it is homozygous or heterozygous. Recessive allele - an allele that is not expressed in the heterozygous state.

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Homozygous - a true breeding organism having two identical alleles for a given characteristic. Heterozygous - having two different alleles for a given characteristic. Phenotype - the expressed (observable) trait or traits of an individual. Genotype - the genomic makeup of an individual. The Punnett-square (named after a British biologist Reginald .C. Punnett) explains the Mendel’s hypotheses at the genotype level. Dominant homozygous Recessive homozygous

AA aa

Hybrid (heterozygous)

Aa

Two types of gametes

A, a

Three genotypes

AA, Aa, aa

Genotypes (1:2:1) AA 25% Aa 50% aa 25%

Phenotypes (3:1) purple 1 purple 2 white 1

Mendel’s principle could be explained on the chromosome basis • Diploid individual, as in pea plant or human, possesses two sets of homologous chromosomes. • Each set comes from male/female parent. • Two chromosomes bear two alleles (the same or different) at the same positions. Mendel’s first law (the law of segregation) - when any individual produces gametes, the alleles separate, so that each gamete receives only one member of the pair of alleles. Two Trait Crosses A genetic cross in which the parents differ with respects to the alleles of two loci of interest, called dihybrid cross. Mendel performed the two trait crosses to see if the two traits of a parent are passed on together in gametes, or whether they segregate independently. Experiments & Results (i.e. seed shape and colour) The phenotypes were:

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P

Round/Yellow seed (♂) X Wrinkled/Green seed (♀) Round/Yellow

F1

In this cross, both parents were homozygous. Traits round and yellow were dominant over wrinkled and green. The genotypes were:

P

RR/YY

X

rryy

RrYy

F1

Mendel considered that in F2, there were two possibilities of the types of gametes produced from RrYy. i. If the two traits passed on together, then only two types of gametes would be formed (RY and ry). In this case, self-fertilization of F1 (RrYy) will yield two phenotypes: 75% round and yellow, and 25% wrinkled and green. ii. The 2nd possibility Mendel considered was that the two traits segregated independent of each other during gamete formation. Thus, 4 types of gametes could be formed in equal numbers ( RY, Ry, rY, ry). When these gametes combine, there is a possibility of 4 types of phenotypes: (round yellow, round green, wrinkled yellow, winkled green) Results: The ratio Mendel obtained was 9:3:3:1. In F2, Mendel got 2 phenotypes similar as original parents, and two new mixed phenotypes appeared. The genotypes of these 4 phenotypes can be predicted using Punnett-square. Nine genotypes were: RRYY, RRYy, RrYY, RrYy, RRyy, Rryy, rrYY, rrYy, rryy (1) (2) (2) (4) (1) (2) (1) (2) (1) Corresponding to four phenotypes: 5

(round yellow, 9

round green, 3

wrinkled yellow, 3

winkled green) 1

In various dihybrid combinations of seven pea traits, Mendel observed that each of two traits produced a 3:1 ratio of phenotypes in the F2, and two trait combination always showed a 9:3:3:1 ratio. Accordingly, Mendel formulated the law of independent assortment (Mendel’s second law) - Alleles of different genes assort independently of one another during gamete formation. Variations of Mendel’s Principles • • • •

Incomplete dominance - a type of inheritance in which F1 hybrids have an intermediate appearance between the phenotypes of the parents. Codominance - a condition in which the phenotypic effects of a gene’s alleles are fully and simultaneously expressed in the heterozygote. Pleiotropy - the determination of more than one character by a single gene. Polygenic inheritance - the additive effect of two or more gene loci on a single phenotypic characteristic.

Testcross A cross of an individual of unknown genotype (which may be either heterozygous or homozygous) for a particular characteristic with a homozygous-recessive individual for that same characteristic.

3 - Chromosomal Basis of Inheritance • Genes are located on chromosomes. • Behavior of chromosomes during meiosis and fertilization accounts for Mendel’s principles of inheritance patterns. After Mendel’s experiments, other workers discovered an inheritance pattern that seemed totally inconsistent with Mendelian principles. In F2 of dihybrid crosses, ratios other than 9:3:3:1 were obtained. Bateson-Punnett’s experiment (1908) Experiments: • Two traits in sweet peas: flower colour and pollen shape. • Cross of heterozygous plants (PpLl) that expressed the dominant traits purple flower (P) and long pollen grains (L) (the corresponding recessive traits are red flower (p) and round pollen (l). Results: Single trait segregated following Mendel’s segregation principle, producing a phenotypic ratio of roughly 3:1.

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Two trait segregation, however, did not produce the 9:3:3:1 ratio that is predicted for a dihybrid cross. Instead, a larger proportion of plants with purple flower and long pollen (284 of 381, ~75%) and red flower and round pollen (55 of 381, ~15%) were observed. Explanation: The factors (genes) for the two traits may not be segregating evenly in all of the gametes formed. In this experiment, meiosis in the heterozygous (PpLl) pea plant produces two predominant types of gametes (PL and pl) rather than equal numbers of the 4 types of gametes formed. The large numbers of plants with purple long and red round traits resulted from reunion (fertilization) among PL and pl gametes. Several years later, it was revealed that the dominant genes for purple colour and long pollen are located on the same parental chromosome. Generally, many genes do not follow Mendel’s principle of independent assortment. • In an organism, each chromosome possesses many genes. o Human genome revealed 26,588 protein-encoding genes, but 23 pairs of chromosomes. o Arabidopsis has 25,498 genes encoding proteins within 5 chromosomes. o Fission yeast contains 4,824 protein-containing genes distributed in 3 chromosomes. • During meiosis, close localized genes on a chromosome tend to be passed on together to a gamete. • Thus, the genes that are located close together on the same chromosome are linked, called linked genes. Question: What about the less numbers of plants with purple round and red long traits (recombinants)? Crossing over and genetic mapping In Bateson-Punnett’s experiments, some Pl and pL gametes were also formed. Crossing over accounts for the recombinant gamete formation and recombines linked genes into assortments of alleles not found in parents. Crossing over is the exchange of segments between chromatids of homologous chromosomes during meiosis. T.H. Morgan and his students (1900s) first demonstrated the effects of crossing over on fruit fly (Drosophila melanogaster) phenotype recombination.

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Using segregation ratios to predict recombination frequencies (percentage of recombinants), linkage maps could be developed to help determine the position of different genes or loci on the chromosome (learn in the lab). Sex Chromosome For many organisms, including human, sex of organisms is determined by one of the chromosomes - the sex chromosome. Different species, the sex chromosomes are different in numbers or kinds. In humans, ♀ has XX and all gametes have X; ♂ has XY and produces 2 types of gametes: X and Y. Therefore, the sex of the offspring is determined by the gametes from ♂. In birds and fishes, ♂ is XX (called ZZ) and ♀ has XY (called ZW). Thus, the situation is opposite of humans: the ♀ (egg) determines the sex of offspring. In some insects such as grasshoppers, ♀ as XX, but ♂ has only one X; Y chromosome is absent. Here ♂ is designated XO. In other insects such as ants and bees, sex is determined by chromosome numbers. ♀ develops from the fertilized egg (diploid); ♂ develops from the unfertilized egg (haploid). Sex-linked inheritance Human individuals with abnormal sex chromosomes could be physically normal, but sterile. • Human XO individuals are females who could be physically moderately abnormal and mentally normal, but sterile (Turner syndrome). • XXY individuals are males who could be physically normal, but always sterile (Klinefelter syndrome). Many of the genes on sex chromosomes are for non-sexual traits. In humans, Y chromosome contains only about 20 known genes, among them are the maleness determinants. However, X chromosome contains a large number of genes. Earlier we learnt that chromosomes exist in homologous pairs in a diploid organism. Males however contain a single X and Y chromosomes. Several important human diseases are inherited as X-linked recessives, including muscular dystrophy, colour blindness and hemophilia which are expressed much more frequently in men than in women. Extranuclear Inheritance (cytoplasmic transmission) Some traits in eukaryotes do not adhere to expected patterns associated with the

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biparental inheritance characteristic of Mendelian genetics. Transmission could be associated with female parent: • Maternal gene products that influence early development. • Mitochondrial and chloroplast genomes that affect the offspring’s phenotype. • Infectious particles.

4 - The Genetic Material Characteristics of the genetic material • Replication • Storage of information • Expression of that information • Variation by mutation Discovery of DNA as the genetic material Historical account: • Early studies had indicated that the genetic material resides in the nucleus. • When nucleus was stained with specific dyes, it was shown to consist of proteins and nucleic acids. Until the 1940s, many geneticists favored proteins: • Abundant • Many forms Evidence favoring DNA: In body cells, the amount of DNA was constant. However, in gametes it was reduced to ½. I -- Prokaryotic bacterial transformation (Frederick Griffith’s experiment, 1927-1928) Two strains of the bacterium, Streptococcus pneumoniae - a bacterium that is one causing agent of the lung disease pneumonia, were used to inject laboratory mice. A smooth(S) strain virulent A rough(R) strain nonvirulent

mice died mice survived

The S strain has a polysaccharide coat that protects bacteria from the host’s defense system. The R strain lacks this coat and will be destroyed by the host defense. In Griffith’s experiments: Heat-killed S bacteria Heat killed S bacteria + R bacteria

injection

mice lived

injection

mice died

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When Griffith examined the blood of the dead mice, they had the S bacteria. It was suggested that R bacteria had been transformed with the genetic material from S bacteria. The genetic material from S bacteria had converted the nonvirulence (avirulent) to the virulence. It was not until 1944 that Oswald Avery and his colleagues at the Rockefeller University identified the substance as DNA. They purified DNA from S bacteria and transferred it to R bacteria which converted R bacteria to be virulent. These experiments first established that DNA is the genetic material in cells. II -- Bacteriophage (bacterial virus) infection (The Hershey-Chase experiments 1952) These experiments conducted by Alfred Hershey and Martha Chase at the Carnegie labs provided much more of convincing evidence. They used a virus T2 that infects bacteria. The virus has a simple structure that consists of an outer protein coat and has the DNA inside the coat. Hershey and Chase fed T2 infected bacteria with radioactive compounds: P32 (phosphorus) and S35 (sulfur) and incorporated them into viral DNA and protein, respectively. When these radiolabeled viruses were used to infect bacteria, the virus DNA, not the proteins, entered the host bacterial cells. The virus attacks the host bacterium, inserts its DNA into the host, where it replicates and takes over the host cell machinery, and produces many more T2 viruses. • •

This supported the view that DNA is the hereditary material and carried over to the next generation, and not the proteins. Results also indicated the DNA contains the information for not only forming the new DNA, but also the new protein coat in new viruses.

DNA and RNA Structures Nucleic acid chemistry Nucleic acids are polymers consisting of many nucleotide monomers, including DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Nucleotide consists of three essential components: a nitrogenous base a pentose (ribose, a five-carbon sugar) a phosphate group

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Two kinds of nitrogenous bases: Purines (Adenine, Guanine) Pyrimidines (Cytosine, Thymine, Uracil) Nucleoside is composed of a nitrogenous base and a ribose/deoxyribose sugar. Nucleotides and nucleosides are named according to the specific nitrogen base (A, C, G, T and U). Nucleotides Nucleosides i.e. (deoxy)adenylic acid (deoxy)adenosine Nucleotides are also described by Nucleoside monophosphate Nucleoside diphosphates Nucleoside triphosphates Polymerization of mononucleotides forms nucleic acid. The linkage between two mononucleotides consists of a phosphate group linked to two sugars forming a phosphodiester bond. DNA Structure • Biochemical analysis of hydrolyzed samples of DNA revealed that the amount of adenine equals the amount of thymine (A=T) and the amount of guanine equals the amount of cytosine (C=G). • X-ray crystallography showed that the DNA molecule is a helix. In 1953, James Watson and Francis Crick proposed that the structure of DNA is in the form of a double-stranded helix. • DNA molecule consists of two strands, running in the opposite direction (antiparallel). • The backbone of strand is sugar-phosphate linked end to end through phosphodiester bonds. • Nitrogenous bases connect the strands in the middle through H-bonds. The bases in two strands are complementary to each other (base pairing). • Two strands are twisted (coiled) to the right. RNA Structure • RNA is generally a single strand polynucleotide although in some cases it may fold upon itself to form internal base pairing. • The sugar molecule is ribose instead of the deoxyribose. • One of the pyrimidines, thymine, is replaced by another nitrogenous base, uracil. Three major types of RNA: • messenger RNA (mRNA) • transfer RNA (tRNA) 11

•

ribosome RNA (rRNA)

DNA – the Hereditary Material • How is the genetic information coded in the DNA molecule? – The information must lie on the linear sequence of nitrogenous bases; i.e. the arrangement of bases. •

How is the information passed on from one cell to the next (i.e. during mitosis)? – It must be because of the complementary base pairing of nitrogenous bases, that one strand serves as a template to form a new strand (DNA replication).

•

How does the genetic material function? It must be that the DNA passes on its information (transcription) where is this used in cell function.

5 - DNA Replication -- the process by which DNA is duplicated (DNA

DNA).

A semi-conservative process in which a double helix gives rise to two double helices, each with an old strand and a newly synthesized strand. Process requires: • “untwisting” of the DNA molecule. This occurs by the breaking of H bonds between N-bases, promoted by certain enzymes. • Each of two strands now severs as a “template” to synthesize a new complementary strand. • A specific enzyme called “DNA polymerase” helps in the linking of free nucleotides to form a new strand. • The nucleotides are always added at the 3’-end (which has a free-OH group) of the new strand. • The template (old) and complementary (new) strands are antiparallel; the 3’end of one strand is paired with 5’-end of the other. • Both parent strands are replicated, and replication proceeds in many locations of each strand forming DNA pieces. • DNA pieces are linked by an enzyme called “DNA ligase”. • The two DNA molecules formed are identical, i.e. in base sequence, to the parent molecule. Each molecule has ½ of the old and ½ new molecule - semiconservative. 6 - The Flow of Genetic Information In this chapter, we explain how cells decode and use the information in their genomes. The Central Dogma is DNA

RNA

Protein

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Transcription -- DNA-directed RNA synthesis (DNA

RNA)

Processes: • Unwinding of the DNA double helix. • In a given region of DNA, only one strand acts as a template. • Initiation of transcription requires “RNA polymerase” binding to a promoter region. • Elongation: RNA polymerase catalyzes the transcription, and U rather than T incorporates into RNA. • Transcription ends at a termination site (terminator). Product: • Transcripts -- RNAs (mRNA, tRNA, rRNA). The Genetic Code -- The basic language of protein synthesis in cells; the genetic code consists of triplets (codons) of nucleotides. Each codon specifies an amino acid in a polypeptide, or a signal to either start or terminate polypeptides synthesis. • Since there are four bases, there are 64 possible codons. • One start codon (ATG) indicates the starting point of translation, and codes for methionine. • Three stop codes indicate the end of translation. • 60 codons code for particular amino acids. Since there are only 20 different amino acids, the genetic code could be redundant for certain amino acids, but the code is not ambiguous. Translation -- the conversion of information provided by mRNA into a specific sequence of amino acids in a polypeptide chain; RNA-directed polypeptide synthesis (RNA Protein) In prokaryotes, transcription and translation both takes place in cytoplasm, while in eukaryotic cells, transcription occurs in nucleus and translation occurs in the cytoplasm. Process requires: • amino acids (20 AA) • RNAs (mRNA, tRNA, rRNA) • Ribosomes (large and small subunits) • enzymes and proteins • ATP Process: • Amino acids are linked in an order specified by the codons in mRNA.

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• • • • • •

It is achieved by an adaptor, tRNA, which binds the correct amino acid catalyzed by an “aminoacyl-tRNA synthetase” and has an anticoden complementary to the mRNA codon. mRNA meets the “charged tRNAs” at a ribosome which consists of a large and a small units and has binding sites for mRNA and tRNAs (“P site” and “A site”). Polypeptides initiate with a methionine and grow from the N-terminus towards the C-terminus along the mRNA in the 5’- to 3’- direction. The large subunit has “peptidyl transferase activity” and catalyzes the reaction. The ribosome moves along the mRNA one codon at a time. The presence of a stop codon in the A site of the ribosome causes translation to be terminated.

Product: Proteins -- the molecular basis of the phenotype (trait) lies in proteins with a variety of functions.

7 - The Control of Gene Expression Gene expression - the flows of genetic information from the genotype to the phenotype (DNA RNA Protein). Gene Expression Regulated in Prokaryotic Cells Operon: A genetic unit of transcription, typically consisting of several structural genes, that are transcribed together, and at least two control (regulatory) regions: the promoter and the operater. Structure gene: A gene that encodes the primary structure of a protein. Promoter: the region of an operon that acts as the initial binding site for RNA polymerase. Operator: the region of an operon that acts as the binding site for the regulatory proteins, usually repressors. Promoters and operators do not code for proteins, but serve as binding sites for regulatory proteins. Regulatory gene: a gene that contains the information for making a regulatory macromolecule, often a repressor protein. Repressor: a protein coded by the regulatory gene that can bind to a specific operator and prevent transcription of the operon. An inducible operator-repressor system: Lactose utilization - Lac Operon

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Three structure genes: β-Galactosidase β-Galactoside permease β-Galactoside transacetylase Transcription is induced by the removal of a repressor A repressible operator-repressor system: Tryptophan biosynthesis - trp Operon Five structural genes. Transcription is repressed by the binding of a repressor. Gene Expression Regulated in Eukaryotic Cells Gene expression regulated at transcriptional level Each eukaryotic gene usually has its own set of control sequences. Regulatory proteins: • Regulatory genes encoding regulatory proteins called transcriptional factors (activators and repressors, and other proteins). • Many more regulatory proteins forming as a complex are involved. • Activator proteins (activator: a protein coded by the regulatory gene that switches on a gene or group of genes) seem to be more important in eukaryotes than repressor. DNA binding sites of activator and repressor: Enhancer: a sequence of eukaryotic DNA, lying on either side of the gene it regulates, that stimulates a specific promoter. Silencer: a sequence of eukaryotic DNA that binds regulatory proteins that inhibit the transcription of an associated gene. Gene expression regulated at post-transcriptional level A typical eukaryotic protein-coding gene has both coding sequences (exons) and non-coding sequences (internal non-coding sequences-intron; flanking sequencescap and tail). exon - a coding portion of a gene. intron - a non-coding portion of a gene that is excised from the RNA transcript. RNA splicing - the removal of introns and joining of exons in eukaryotic RNA, forming an mRNA molecule with a continuous coding sequence. RNA splicing occurs in a spliceosome (a large RNA-protein complex) before mRNA. • Alternate splicing could produce different proteins. • Flanking sequences could regulate the stability of mRNA in the cytoplasm.

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Biological functions of introns: • Alternate splicing generates different mRNA molecules from the same RNA transcript. • Intron may also contribute to genetic diversity. Gene expression regulated at translation and post-translation levels • Translation machinery involves many proteins that have regulatory functions. • Protein cleavage/modification/activation. • Targeted protein degradation through proteasomes.

8 - Biotechnology GMO - an organism that has acquired the genetic material by artificial means. In agricultural application: • Insecticide production in plant • Resistance to herbicides • Improved nutritional characteristics Stem Cells - a relatively unspecialized cell that can give rise to other specialized cells.

9 - The Genome Era Genomics Bioinformatics

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