AP Biology Notes Outline Enduring Understanding 3.A

L. Carnes

Big Idea 3: Living systems store, retrieve, transmit and respond to information essential to life processes.

Enduring Understanding 3.A:

Heritable information provides for continuity of life.

Learning Objectives: Essential Knowledge 3.A.1: DNA, and in some cases RNA, is the primary source of heritable information.

– (3.1) The student is able to construct scientific explanations that use the structures and mechanisms of DNA and RNA to support the claim that DNA and, in some cases, that RNA are the primary sources of heritable information.

– (3.2) The student is able to justify the selection of data from historical investigations that support the claim that DNA is the source of heritable information.

– (3.3) The student is able to describe representations and models that illustrate how genetic information is copied for transmission between generations. – (3.4) The student is able to describe representations and models illustrating how genetic information is translated into polypeptides. – (3.5) The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly used technologies. – (3.6) The student can predict how change in a specific DNA or RNA sequence can result in changes in gene expression.

Essential Knowledge 3.A.2: In eukaryotes, heritable information is passed to the next generation via processes that include the cell cycle and mitosis or meiosis plus fertilization.

– (3.7) The student can make predictions about natural phenomena occurring during the cell cycle. – (3.8) The student can describe the events that occur in the cell cycle. – (3.9) The student is able to construct an explanation, using visual representations or narratives, as to how DNA in chromosomes is transmitted to the next

generation via mitosis, or meiosis followed by fertilization. – (3.10) The student is able to represent the connection between meiosis and increased genetic diversity necessary for evolution. – (3.11) The student is able to evaluate evidence provided by data sets to support the claim that heritable information is passed from one generation to

another through mitosis, or meiosis followed by fertilization.

Essential Knowledge 3.A.3: The chromosomal basis of inheritance provides an understanding of the pattern of passage (transmission) of genes from parent to offspring.

– (3.12) The student is able to construct a representation that connects the process of meiosis to the passage of traits from parent to offspring. – (3.13) The student is able to pose questions about ethical, social or medical issues surrounding human genetic disorders. – (3.14) The student is able to apply mathematical routines to determine Mendelian patterns of inheritance provided by data sets.

Essential Knowledge 3.A.4: The inheritance pattern of many traits cannot be explained by simple Mendelian genetics.

– (3.15) The student is able to explain deviations from Mendel’s model of the inheritance of traits. – (3.16) The student is able to explain how the inheritance patterns of many traits cannot be accounted for by Mendelian genetics. – (3.17) The student is able to describe representations of an appropriate example of inheritance patterns that cannot be explained by Mendel’s model of the

inheritance of traits.

Bozeman Instruction Videos: http://www.bozemanscience.com/ap-biology/ 3.A.1 - #027: DNA & RNA Part I 3.A.1 - #027: DNA & RNA Part II 3.A.2 - #028: Cell Cycle, Mitosis & Meiosis 3.A.3 - #029: Mendelian Genetics 3.A.4 - #030: Advanced Genetics

Required Readings: 3.A.1 - Textbook Ch. 16; 17; and 20 (pp. 396-399; pp. 405-406; pp. 412-422) 3.A.2 - Textbook Ch. 12 and 13 3.A.3 - Textbook Ch. 14 3.A.4 – Textbook Ch. 15

Practicing Biology Homework Questions: Questions #1-20

Essential Knowledge 3.A.1: DNA, and in some cases RNA, is the primary source of heritable information.

The organizational basis of all living systems is heritable information. The proper storage and transfer of this information is critical for life to continue at the cell, organisms and species level. Reproduction occurs at the cellular and organismal levels. In order for daughter cells to continue subsequent generational cycles of reproduction or replication, each progeny needs to receive heritable genetic instructions from the parental source. This information is stored and passed to the subsequent generation via DNA. Viruses, as exceptional entities, can contain either DNA or RNA as heritable information. The chemical structures of both DNA and RNA provide mechanisms that ensure information is preserved and passed to subsequent generations. There are important chemical and structural differences between DNA and RNA that result in different stabilities and modes of replication. In order for information stored in DNA to direct cellular processes, the information needs to be transcribed (DNARNA) and in many cases, translated (RNAprotein). The products of these processes determine metabolism and cellular activities and, thus, the phenotypes upon which evolution operates. Genetic information is stored and transmitted from one generation to the next through DNA or RNA. Different types of organisms have different chromosome structure. Prokaryotic organisms have circular chromosomes, while eukaryotic organisms have multiple linear chromosomes, although in biology there are exceptions to this rule. The proof that DNA is the carrier of genetic information involved a number of important historical experiments. Notable experiments include: 1. Morgan’s experiments on inheritance patterns in Drosophila melanogaster 2. Griffith’s experiments on transforming bacteria 3. Avery-McLeod-McCarty experiments 4. Hershey-Chase experiment 5. Contributions of Watson, Crick, Wilkins, and Franklin on the structure of DNA Early in the 20th century, the identification of the molecules of inheritance loomed as a major challenge to biologists. • When T. H. Morgan’s group showed that genes are located on chromosomes, the two components of chromosomes— DNA and

protein —became candidates for the genetic material. • The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect them. Figure 16.2: Evidence that Bacteria Can Be Transformed – GRIFFITH’S TRANSFORMATION EXPERIMENTS http://nortonbooks.com/college/biology/animations/ch12a01.htm • The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928. Griffith worked with two strains of a

bacterium, one pathogenic and one harmless. • The S-strain of Streptococcus pneumoniae causes pneumonia; the R-strain is nonpathogenic. Griffith discovered that a mixture

of heat-killed S cells and live R cells killed mice and that live S bacteria could be retrieved from the dead mice. • He concluded that molecules from the dead S cells had genetically transformed living R bacteria into S bacteria. When he mixed

heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic. He called this phenomenon transformation, and concluded that molecules from the dead S cells had genetically transformed living R bacteria into S bacteria.

• Problem: what factor is causing this transformation? Avery, McCarty and MacLeod Experiments http://www.sinauer.com/cooper5e/animation0401.html • In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance from Griffith’s

experiments was DNA. These scientists focused on three main candidates – DNA, RNA, and protein. Avery broke open the heat-killed pathogenic bacteria and extracted cellular contents.

• In separate samples, he used specific treatments that inactivated each of the three types of molecules. He then tested each treated sample for its ability to transform live nonpathogenic bacteria.

• Only when DNA was allowed to remain active did transformation occur. • Many biologists remained skeptical, mainly because little was known about DNA.

Fig. 16-4: The Hershey Chase Experiment http://nortonbooks.com/college/biology/animations/ch12a02.htm • In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of cells. They

used bacteriophages to answer this question – these are viruses that infect bacteria. Bacteriophages are made of only two components – DNA and protein.

• They used a radioactive isotope of phosphorus to tag the DNA in one culture of bacteriophages and radioactive sulfur to tag the protein in a second culture. There results clearly showed that only the DNA entered bacteria infected by the virus; the radioactive protein never entered the cell. THIS RESEARCH CONVINCED MANY SCIENTISTS THAT DNA MUST BE THE GENETIC MATERIAL OF CELLS.

• The Hershey-Chase experiment was a landmark study because it provided powerful evidence that nucleic acids, rather than proteins, are the hereditary material, at least for viruses.

Additional Evidence that DNA is the Genetic Material of Cells • Further evidence that DNA is the genetic material of cells came from the laboratory of biochemist Erwin Chargaff. It was

already known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group. • In 1950, Chargaff reported that DNA composition varies from one species to the next after analyzing the base composition of

DNA from a number of different organisms. This evidence of diversity made DNA a more credible candidate for the genetic material.

• Chargaff also noticed a peculiar regularity in the ratios of nucleotide bases within a single species. Chargaff’s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases. The basis for these rules, however, remained unexplained until the discovery of the double helix structure of DNA.

Building a Structural Model of DNA • After most biologists became convinced that DNA was the genetic material, the challenge was to determine how its structure

accounts for its role. Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure.

• Franklin produced a picture of the DNA molecule using this technique. Images produced by X-ray crystallography are not actually pictures of molecules…the spots and smudges are produce by X-rays that were diffracted as they passed through aligned fibers of purified DNA.

• Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce that DNA was helical. The X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases. The width suggested that the DNA molecule was made up of two strands, forming a double helix.

• Francis Watson and James Crick built models of a double helix to conform to the X-rays and chemistry of DNA. Franklin had concluded that there were two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior. In 1953, James Watson and Francis Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA. DNA, the substance of inheritance, is the most celebrated molecule of our time. Hereditary information is encoded in DNA and reproduced in all cells of the body. This DNA program directs the development of biochemical, anatomical, physiological, and (to some extent) behavioral traits.

Each nucleotide monomer consists of a nitrogenous base (A, T, C, or G), the sugar deoxyribose, and a phosphate group. The phosphate of one nucleotide is attached to the sugar of the next, resulting in a “backbone” of alternating phosphates and sugars from which the bases project. The polynucleotide strand has directionality, from the 5’ end (with the phosphate group) to the 3’ end (with the –OH group). The 5’ and 3’ refer to the numbers assigned to the carbons in the sugar ring. Thus, the two strands of the DNA double helix are antiparallel, meaning that the 5’ to 3’ direction of one strand runs counter to the 5’ to 3’ direction of the other strand.

At first, Watson and Crick thought the bases paired like with like (A with A, and so on), but such pairings did not result in a uniform width. Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray. A and G are purines (bases with two organic rings) and C and T are pyrimidines (bases with a single organic ring). Watson and Crick reasoned that the pairing was more specific, dictated by the base structures. They determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C). The Watson-Crick model explains Chargaff’s rules: in any organism the amount of A = T, and the amount of G = C. The pairs of nitrogenous bases in a DNA double helix are held together by hydrogen bonds…as shown here.

The two DNA strands are ANTIPARALLEL – that is, their sugar-phosphate backbones run in opposite directions. The 5’ → 3’ direction of one strand runs counter to the 5’ → 3’ direction of the other strand. Notice in the figure that a nucleotide’s phosphate group is attached to the 5’ carbon of deoxyribose. Notice also that the phosphate group of one nucleotide is joined to the 3’ carbon of the adjacent nucleotide. The numbers assigned to the carbon atoms of the deoxyribose are shown for two of them. In the figure, the five carbons of one deoxyribose sugar of each DNA strand are numbered from 1’ to 5’.

DNA replication ensures continuity of hereditary information. • Replication is a semiconservative process; that is, one strand serves as the template for a new, complementary strand. • Replication requires DNA polymerase plus many other essential cellular enzymes, occurs bidirectionally, and differs in the

production of the leading and lagging strands.

The Basic Principle: Base Pairing to a Template Strand The relationship between structure and function is manifest in the double helix:

• Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material. • Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication. • In DNA replication, the parent molecule unwinds, and two new daughter strands are built following the rules of

complimentary base pairing. Figure 16.9: Replication is a Semi-Conservative Process http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html • Watson and Crick’s semiconservative model of replication predicts that when a

double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand.

• Competing models were the conservative model (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new).

• REMEMBER: this all occurs during the S phase of interphase in mitosis –or- during MEIOSIS I ONLY!

• SEMI-CONSERVATIVE: that is, one strand serves as a template for a new, complimentary strand.

http://bcs.whfreeman.com/thelifewire/content/chp11/1102002.html http://highered.mcgraw-hill.com/olc/dl/120076/bio23.swf DNA Replication: A Closer Look http://highered.mcgraw-hill.com/sites/0072943696/student_view0/chapter3/animation__dna_replication__quiz_1_.html • Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a

replication “bubble”. • A eukaryotic chromosome may have hundreds or even thousands of origins of replication. Replication proceeds in both

directions from each origin, until the entire molecule is copied. More than a dozen enzymes and other proteins participate in DNA replication.

• At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating. • Helicases are enzymes that untwist the double helix at the replication forks. Single-strand binding protein binds to and

stabilizes single-stranded DNA until it can be used as a template. RNA primers lay down a short stretch of RNA from which DNA can be extended.

• Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands. • DNA polymerase cannot initiate a polynucleotide strand; it can only add to the 3’ end of an already-started strand. The initial

nucleotide strand is s short RNA Primer. The primer is a short segment of RNA synthesized by the enzyme primase. Primase can start an RNA strand from scratch and adds RNA nucleotides one at a time using the parental DNA as a template.

• The primer is short (5–10 nucleotides long), and the 3 end serves as the starting point for the new DNA strand. Each primer is eventually replaced by DNA.

The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication:

• DNA polymerases add nucleotides only to the free 3 end of a growing strand;

therefore, a new DNA strand can elongate only in the 5 to 3 direction. • Along one template strand of DNA, the DNA polymerase synthesizes a leading strand

continuously, moving toward the replication fork. • Along the other template strand of DNA, the DNA polymerase synthesize a lagging

strand discontinuously in segments called Okazaki fragments, moving away from the replication fork.

http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html

1. During DNA replication, the DNA molecule (1) separates into two strands; (2) produces two complimentary strands following the rules of base pairing - each strand of the double helix serves as a template for a new strand.

2. Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble”. A eukaryotic

chromosome may have hundreds or even thousands of origins of replication. Replication proceeds in both directions from each origin, until the entire molecule is copied.

3. The enzyme helicase unwinds the DNA helix and the enzyme gyrase temporarily breaks the strands of DNA. As the strands are unwound, an area called a replication fork is created. Single-stranded binding proteins (SSBs) assist in preventing the open strands from re-annealing. Topoisomerases relieve “overwinding” strain ahead of the replication forks by breaking, swiveling, and rejoining the DNA strands.

4. DNA polymerase cannot initiate a polynucleotide strand; it can only add to the 3’ end of an already-started strand. The enzyme primase synthesizes a short RNA primer to which DNA polymerase adds new nucleotides to the growing DNA strand by hooking the 5’ phosphate group of an incoming nucleotide onto the 3’ hydroxyl group at the end of the growing nucleic acid chain. Because the chain grows by extension off the 3’ hydroxyl group, strand synthesis is said to proceed in a 5’ to 3’ direction.

5. The primer is a short segment of RNA synthesized by the enzyme primase. Each primer is eventually replaced by DNA.

6. Replication occurs in the 5’ to 3’ direction only – so the two new strands grow in opposite directions. The two DNA strands are ANTIPARALLEL – that is, their

sugar-phosphate backbones run in opposite directions. The 5’ → 3’ direction of one strand runs counter to the 5’ → 3’ direction of the other strand. One strand is referred to as the leading strand; the other is referred to as the lagging strand.

7. The LEADING strand is synthesized continuously toward the replication fork; the LAGGING strand is synthesized discontinuously away from the replication fork (in pieces called Okazaki fragments). DNA ligase seals the gaps between the Okazaki fragments on the lagging strand.

8. Replication is complete when the replication fork reaches the end of the parent strand. The original parent strand and new daughter strand wind into a helix.

Because each strand now has ½ old and ½ new, DNA replication is said to be semiconservative.

Proofreading and Repairing DNA http://nortonbooks.com/college/biology/animations/ch12a05.htm • DURING the replication process, DNA polymerases proofread newly made DNA,

replacing any incorrect nucleotides. • BUT - some incorrect pairs can evade the proofreading mechanism, or arise after

replication and these must be correct AFTER replication is complete.

• In mismatch repair of DNA, repair enzymes correct errors in base pairing • In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches

of DNA

• Mismatch repair enzymes are incredibly important and researchers have found that a hereditary defect in one of them is associated with a form of colon cancer:

• Apparently, this defect allows cancer-causing errors to accumulate in the DNA at a faster rate than normal.

A team of enzymes detects and repairs damaged DNA in nucleotide excision repair. Repair enzymes (nucelases) can excise damaged DNA regions from the DNA and replace them with a normal segment. Good Animation: http://nortonbooks.com/college/biology/animations/ch12a05.htm Viral replication differs from other reproductive strategies. • In living organisms, the flow of genetic information is from DNA to

RNA, and translated into proteins. Replication involves copying a template strand of DNA into a complementary new strand of DNA.

• Genetic information in retroviruses is a special case and has an alternate flow of information: from RNA to DNA, made possible by reverse transcriptase, and enzyme that copies the viral RNA genome into DNA.

• This DNA is then integrated into the host genome and becomes transcribed and translated for the assembly of new viral progeny.

• We will return to this concept while discussing 3.C.3.

DNA and RNA molecules have structural similarities and differences that define function. Both have three components – sugar, phosphate and a nitrogenous base – which form nucleotide units that are connected by covalent bonds to form a linear molecule with 3’ and 5’ ends, with the nitrogenous bases perpendicular to the sugar-phosphate backbone. The basic structural differences include:

• DNA contains deoxyribose while RNA contains ribose. • RNA contains uracil in lieu of thymine in DNA. • DNA is usually double stranded, RNA is usually single stranded. • The two DNA strands in double-stranded DNA are antiparallel in

directionality. Both DNA and RNA exhibit specific nucleotide base pairing that is conserved through evolution: adenine pairs with thymine or uracil, and cytosine pairs with guanine. Purines (G and A) have a double ring structure. Pyrimidines (C, T and U) have a single ring structure.

The sequence of the RNA bases, together with the structure of the RNA molecule, determines RNA function. http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a1.html TYPES OF RNA: 1. Messenger RNA (mRNA) – carries information from DNA in

the nucleus to the ribosomes where the proteins are assembled. It is a partial copy of ONLY the information needed for that specific job. It is read 3 bases at a time – codon.

2. Ribosomal RNA (rRNA) – found in ribosomes and helps in the attachment of mRNA and in the assembly of proteins.

3. Transfer RNA (tRNA) – transfers the needed amino acids from the cytoplasm to the ribosome so the proteins dictated by the mRNA can be assembled. (The three exposed bases are complementary to the mRNA and are called the anticodon).

4. The role of RNAi includes regulation of gene expression at the level of mRNA transcription.

Overview: The Flow of Genetic Information The information content of DNA is in the form of specific sequences of nucleotides. The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins. Genetic information flows from a sequence of nucleotides in a gene (DNA) to a sequence of amino acids for a trait (protein). PROTEINS ARE THE LINKS BETWEEN GENOTYPE AND PHENOTYPE. Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation. Genetic information flows from a sequence of nucleotides in a gene to a sequence of amino acids in a protein. • The process begins when the enzyme RNA-polymerase reads the DNA molecule in the 3’ to 5’ direction and synthesizes

complementary mRNA molecules that determine the order of amino acids in the polypeptide. • In eukaryotic cells the mRNA transcript undergoes a series of enzyme-regulated modifications before being sent to the ribosome

to build the polypeptide: (1) addition of a poly-A tail; (2) addition of a GTP cap; and (3) excision of introns. • In prokaryotic organisms, transcription is coupled to translation of the message. Translation involves energy and many steps,

including initiation, elongation and termination. Prokaryotic v. Eukaryotic Gene Expression http://highered.mcgraw-hill.com/olc/dl/120077/bio25.swf Protein Synthesis is similar in prokaryotes and eukaryotes. In prokaryotes, transcription and translation both occur in the cytoplasm and they occur generally at the same time. In eukaryotes, transcription occurs in the nucleus and translation occurs in the cytoplasm. The mRNA is processed before being sent to the cytosol for synthesis of the protein. Processing of pre-mRNA includes: (1) addition of a poly-A tail; (2) addition of a GTP cap; and (3) excision of introns.

Basic Principles of Transcription and Translation Genes contain the blueprints for building proteins in cells…RNA is the bridge between genes and the proteins for which they code:

Transcription is the synthesis of RNA under the direction of DNA. Transcription produces messenger RNA (mRNA).

Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA.

Ribosomes are the sites of translation.

A primary transcript is the initial RNA transcript from any gene.

The central dogma is the concept that cells are governed by a

cellular chain of command: DNA RNA protein.

The “genetic code” is the sequence of bases in DNA that will code for specific amino acids in a growing polypeptide (protein).

There are 20 possible amino acids. The flow of information from gene to protein is based on a triplet code: a series of non-overlapping, three-nucleotide words.

• These triplets are the smallest units of uniform length that can code for all the amino acids. • Example: AGT at a particular position on a DNA strand results in the placement of the amino acid serine at the

corresponding position of the polypeptide to be produced. Figure 17.4 The Triplet Code

1. During transcription, one of the two DNA strands called the template strand provides a template for ordering the sequence of nucleotides in an RNA transcript

2. During translation, the mRNA base triplets, called codons, are read in

the 5 to 3 direction 3. Each codon specifies the amino acid to be placed at the corresponding

position along a polypeptide 4. Codons along an mRNA molecule are read by translation machinery in

the 5 to 3 direction 5. Each codon specifies the addition of one of 20 amino acids

All 64 codons were deciphered by the mid-1960s. Of the 64 triplets, 61 code for amino acids; 3 triplets are “stop” signals to end translation. The genetic code is redundant but not ambiguous; no codon specifies more than one amino acid…but a particular amino acid can be specified by more than one codon. Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced.

Transcription: Converting DNA to mRNA http://bcs.whfreeman.com/thelifewire/content/chp12/1202001.html During transcription, RNA polymerase uses one strand of DNA as a template to assemble nucleotides into a strand of RNA. Transcription, the first stage of gene expression, is the DNA-directed synthesis of mRNA.

1. INITIATION - After RNA polymerase binds to the promoter, the DNA strands unwind, and the enzyme initiates RNA synthesis at the start point on the template strand.

2. ELONGATION – The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5’ → 3’. In the wake of transcription, the DNA strands re-form a double helix.

3. TERMINATION – Eventually, the polymerases transcribes a terminator sequence, which signals the end of the transcription unit. Shortly thereafter, the RNA transcript is released, and the polymerase detaches from the DNA.

Promoter: sequence in DNA where RNA polymerase attaches and initiates transcription – signals the initiation of transcription Terminator: sequence that signals the end of transcription Transcription Unit: stretch of DNA that is being copied into mRNA RNA Transcript: stretch of mRNA created using DNA as a template. RNA Modification Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm. During RNA processing, both ends of the primary transcript are usually altered. Also, usually some interior parts of the molecule are cut out, and the other parts spliced together. Each end of a pre-mRNA molecule is modified in a particular way:

• The 5 end receives a modified nucleotide 5 cap

• The 3 end gets a poly-A tail • These modifications share several functions: they seem to

facilitate the export of mRNA. They protect mRNA from

hydrolytic enzymes. They help ribosomes attach to the 5 end. The leader and trailer are not translated, nor is the poly(A) tail.

Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions. These noncoding regions are called intervening sequences, or introns. The other regions are called exons because they are eventually expressed, usually translated into amino acid sequences. RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence.

The Functional and Evolutionary Importance of Introns: Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing. Such variations are called alternative RNA splicing. Because of alternative splicing, the number of different proteins an organism can produce is much greater than its number of genes.

Translation: Using an mRNA Template to Construct a Polypeptide http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis Translation is the RNA-directed synthesis of a polypeptide. Generally: is the reading of the codons on the mRNA strand and the sequencing of them into an amino acid sequence – polypeptide. The Players:

• mRNA: already processed within the nucleus via transcription, will be the template for the sequence of amino acids.

• tRNA: transfers amino acids from the cytoplasm to the ribosome. • rRNA (ribosome): adds amino acids together from the tRNA and in the sequence of

the mRNA.

Figure 17.14 The structure of transfer RNA (tRNA) Function: Pick up designated amino acids in the cystol. Deposit the amino acid at the ribosome. Return to the cystol to pick up another amino acid. Structure: It is made out of about 80 nucleotides. It is folded in on its self in a T shape. One important component is called the anti codon. This base pairs with the codon on the mRNA strand.

Figure 17.16 Anatomy of a Functioning Ribosome Function: Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis. Structure: The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA) Each ribosome has a binding site for mRNA and three binding sites for tRNA molecules.

1. The P site holds the tRNA that carries the growing polypeptide chain 2. The A site holds the tRNA that carries the next amino acid to be added to the

chain 3. The E site is the exit site, where discharged tRNAs leave the ribosome

Three Stages of Translation: 1. Initiation (requires energy “GTP”) – SEE FIGURE 17.17

The initiation stage of translation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits. First, a small ribosomal subunit binds with mRNA and a special initiator tRNA. Then the small subunit moves along the mRNA until it reaches the start codon (AUG). Proteins called initiation factors bring in the large subunit that completes the translation initiation complex.

2. Elongation (requires energy “GTP”) – SEE FIGURE 17.18

During the elongation stage, amino acids are added one by one to the preceding amino acid. Each addition involves proteins called elongation factors and occurs in three steps:

Codon Recognition - The mRNA codon in the A site of the ribosome forms a H-bond with the anticodon of an incoming molecule of tRNA carrying its appropriate amino acid. This requires energy. (GTP) (A site)

Peptide Bond Formation - The ribosome catalyzes the formation of a peptide bond between the new amino acid and the growing polypeptide. Catalyzed by the ribosome.

Translocation - The tRNA in the P site and A site are now moved to the E site and P site respectively. The tRNA in the E site will detach and a new codon is open. The ribosome shifts the mRNA by one codon “reading it”. This step requires energy. (GTP)

3. Termination – SEE FIGURE 17.19

Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome. The A site accepts a protein called a release factor. The release factor causes the addition of a water molecule instead of an amino acid. This reaction releases the polypeptide, and the translation assembly then comes apart

Phenotypes are determined through protein activities.

Genetic engineering techniques can manipulate the heritable information of DNA and, in special cases, RNA. Genetic engineering is the process of manipulating genes and genomes.

• Recombinant DNA is DNA that has been artificially made, using DNA from different sources – and often different species. • Gene cloning is the process of producing multiple copies of specific segments of DNA. • Restriction enzymes are used to cut strands of DNA at specific locations (called restriction sites).

Using Restriction Enzymes to Cut DNA http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf • Restriction enzymes are used to cut strands of DNA at specific locations (called

restriction sites). They are derived from bacteria. • When a DNA molecule is cut by restriction enzymes, the result will always be a set of

restriction fragments, which will have at least one single-stranded end, called a sticky end.

• Sticky ends can form hydrogen bonds with complementary single-stranded pieces of DNA. These unions can be sealed with the enzyme DNA ligase.

Plasmid-Based Transformation http://highered.mcgraw-hill.com/olc/dl/120078/micro10.swf 1) Identify and isolate the gene of interest and a cloning vector. The vector will carry the DNA sequence to be cloned and is often a

bacterial plasmid. 2) Cut both the gene of interest and the vector with the same restriction enzyme. This gives the plasmid and the gene of interest

matching sticky ends. 3) Join the two pieces of DNA. Form recombinant plasmids by mixing the plasmids with the DNA fragments. The DNA from the

gene of interest can be sealed into the plasmid using DNA ligase. 4) Get the vector carrying the gene of interest into the host cell. The plasmids are taken up by the bacterium by transformation. 5) Select for cells that have been transformed. The bacterial cells carrying the clones must be identified or selected. This can be

done by linking the gene of interest to an antibiotic resistance gene or a reporter gene such as green fluorescent protein. Any bacterial cells that do not pick up the plasmid will not grow on agar with antibiotic on it, nor will they express green fluorescence.

Using Gel Electrophoresis to Separate DNA http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html • One indirect method of rapidly analyzing and comparing genomes is gel

electrophoresis. This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size.

• A current is applied that causes charged molecules to move through the gel. • Molecules are sorted into “bands” by their size. • The DNA is loaded into shallow wells at the negative end of the apparatus. An

electrical charge is applied. • The negative charges on phosphates in the molecule cause the DNA to move

toward the positive pole of the electrophoresis apparatus. • The gel allows smaller molecules to move more easily than larger fragments of

DNA, therefore, the smaller fragments move farther on the gel.

Figure 20.10: Using Restriction Fragment Analysis to Distinguish the Normal and Sickle-cell Alleles of the β-globin Gene In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis. Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene In the example

(a) the sickle-cell mutation destroys one of the DdeI restriction sites within the gene, so (b) as a result, digestion with the DdeI enzyme generates different fragments from the normal and sickle-cell alleles.

The products of genetic engineering include, but are not limited to, GM foods; transgenic animals; cloned animals; and pharmaceuticals. • Biotechnology is the process of manipulating organisms of their components for the purpose of making useful products.

Illustrative examples include: – Genetically modified foods; – Transgenic animals; – Cloned animals; – Pharmaceuticals, such as human insulin

Genetically modified foods are foods produced from organisms that have had specific changes introduced into their DNA using the methods of genetic engineering. These techniques have allowed for the introduction of new crop traits as well as a far greater control over a food's genetic structure than previously afforded by methods. Benefits of GM Foods include:

– Ensuring an adequate food supply for the growing human population; – Pest resistance or herbicide tolerance reduces the need for harmful chemicals; – Disease resistance and economics; – Cold/drought tolerance; – Nutritional value of commonly consumed foods. – To date most genetic modification of foods have primarily focused on cash crops in high demand by farmers such

as soybean, corn, canola, and cotton seed oil. These have been engineered for resistance to pathogens and

herbicides and better nutrient profiles. GM livestock have also been experimentally developed, although as of

November 2013 none are currently on the market. There is broad scientific consensus that food on the market

derived from GM crops poses no greater risk to human health than conventional food. However, opponents have

objected to GM foods on several grounds, including safety issues, environmental concerns,

and economic concerns. Some are concerned about the creation of “super weeds” from the transfer of genes

from GM crops to their wild relatives.

Transgenic animals are made by introducing genes from one species into the genome of another animal. Transgenic animals are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use. “Pharm” plants are also being developed to make human proteins for medical use. http://www.learner.org/courses/biology/archive/animations/hires/a_gmo3_h.html A transgenic goat carries a gene for human blood protein, antithrombin, which she secretes in her milk. Patients with a rare hereditary disorder in which this protein is lacking suffer from formation of blood clots in their vessels. Easily purified from the goat’s milk, the protein is currently under evaluation as an anticlotting agent.

In animal cloning the nucleus of an egg is removed and replaced with the diploid nucleus of a body cell. • The major goal of most animal cloning is reproduction, but not for humans. In humans, the major goal is the production of

stem cells. • A stem cell can both reproduce itself indefinitely and, under the proper conditions, produce other specialized cells. • Stem cells have enormous potential for medical application. Embryonic stem cells are capable of differentiating into many

cell types. • The ultimate aim is to use them for the repair of damaged or disease organs, such as insulin-producing pancreatic cells for

people with diabetes or certain kinds of brain cells for people with Parkinson’s disease. Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases. Host cells in culture can be engineered to secrete a protein as it is made. This is useful for the production of insulin, human growth hormones, and vaccines. Among the first pharmaceutical products “manufactured” were human insulin. Some 2 million people with diabetes in the US depend on insulin treatment to control their disease. Insulin from cows and pigs has been used since the early 1900s to treat diabetes. Now human insulin protein can be mass-produced through genetic engineering processes.

1. Isolate Gene - The gene for producing HUMAN insulin protein is isolated. The gene is part of the DNA in a human chromosome. The gene can be isolated and then copied so that many insulin genes are available to work with. 2. Prepare Target DNA - In 1973, two scientists named Boyer and Cohen developed a way to take DNA from one organism and put it in the DNA of bacterium. This process is called recombinant DNA technology. First, a circular piece of DNA called a plasmid is removed from a bacterial cell. Special proteins are used to cut the plasmid ring open. 3. Insert DNA into Plasmid - With the plasmid ring open, the gene for insulin is inserted into the plasmid ring and the ring is closed. The human insulin gene is now recombined with the bacterial DNA plasmid. 4. Insert Plasmid back into cell - The bacterial DNA now contains the human insulin gene and is inserted into a bacteria. Scientists use very small needle syringes to move the recombined plasmid through the bacterial cell membrane. 5. Plasmid multiply - Many plasmids with the insulin gene are inserted into many bacterial cells. The cells need nutrients in order to grow, divide, and live. While they live, the bacterial cell processes turn on the gene for human insulin and the insulin is produced in the cell. When the bacterial cells reproduce by dividing, the human insulin gene is also reproduced in the newly created cells. 6. Target Cells Reproduce - Human insulin protein molecules produced by bacteria are gathered and purified. The process of purifying and producing cow and pig insulin has been greatly reduced or eliminated. 7. Cells Produce Proteins - Millions of people with diabetes now take human insulin produced by bacteria or yeast (biosynthetic insulin) that is genetically compatible with their bodies, just like the perfect insulin produced naturally in your body.

Essential Knowledge 3.A.2: In eukaryotes, heritable information is passed to the next generation via processes that include the cell cycle and mitosis or meiosis plus fertilization.

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Essential Knowledge 3.A.3: The chromosomal basis of inheritance provides an understanding of the pattern of passage (transmission) of genes from parent to offspring.

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Essential Knowledge 3.A.4: The inheritance pattern of many traits cannot be explained by simple Mendelian genetics.

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