(1) Chapter title: The Molecular Basis of Inheritance (a) [the molecular basis inheritance (Google Search)] [index]

(2) Chromosomes (a) Chromosomes consist of DNA and protein (b) Which is the hereditary material? The history of our understanding is outlined in the efforts of the following: (i) Griffith, 1928 (ii) Avery, MacLeod, and McCarty, 1944 (iii) Hershey and Chase, 1952 (iv) Chargaff (v) Franklin and Wilkins (vi) Watson and Crick, 1953 (c) [chromosomes (Google Search)] [index] (3) Griffith, 1928 (a) Griffith discovered a hereditary molecule that was transmittable between bacteria (b) See Figure 16.1, Transformation of bacteria (c) [Griffith 1928 (Google Search)] [index] (4) Avery, MacLeod, and McCarty, 1944 (a) Avery et al. found that Griffiths transmittable hereditary molecule is DNA (b) In particular, they showed that the transmittable hereditary molecule was susceptible to the DNA hydrolyzing enzyme known generically as DNAse (c) [Avery MacLeod McCarty, DNAse OR DNAase, DNAse, DNAase (Google Search)] [index] (5) Hershey and Chase, 1952 (a) Hershey and Chase showed that the hereditary material in T2 bacteriophages is DNA, thereby generalizing Avery, MacLeod, and McCartys observation (b) See Figure 16.2, The Hershey-Chase experiment (c) [Hershey Chase, Alfred Day Hershey (Google Search)] [the bacteriophage ecology group (Microdude)] [index] (6) Chargaff (Chargaffs rule) (a) Chargaff found that different species of organisms have different DNA nucleotide compositions (b) Chargaffs rule states that the fraction of nucleotides that makes up an organisms DNA always behaves the rule: the fraction of As = the fraction of Ts, and the fraction of Gs = the fraction of Cs (i.e., the fractions of A + T + G + C = 1; A = T and G = C; 2 * (A + G) = 1, etc.) (c) [Chargaff, Chargaff's rule (Google Search)] [index] (7) Franklin and Wilkins (a) Franklin and Wilkins are responsible for supplying an X-ray diffraction of DNA, essentially an in-this-case crude molecular picture of the molecule, that indicated the basic structural features that DNA possesses: (i) The periodicity of DNA (ii) The molecules uniform width

(iii) That the nitrogenous bases stacked 0.34 nm apart (b) See Figure 16.4, Rosalind Franklin and her X-ray diffraction photo of DNA (c) [Franklin Wilkins DNA, DNA X-ray diffraction (Google Search)] [index] (8) Watson and Crick, 1953 (a) Watson and Crick, in 1953, published the double helix model of DNAs structure (b) J. D. Watson and F. H. C. Crick (1953). Molecular Structure of Nucleic Acids. Nature, vol. 171 (25 April 1953), pages 737-738 (c) This paper was, arguably, the single most important contribution to biology (and perhaps even chemistry as well) of the twentieth century (d) See Figure 16.5, The double helix (e) The Watson and Crick model: (i) Explains DNAs periodicity (ii) Explains DNAs uniform width (iii) Explains Chargaffs rule (iv) Explains how DNA is replicated (f) (you will not be held responsible for the above history) (g) [Watson Crick, double helix (Google Search)] [annotated version of Watson & Crick, 1953!, (nice index of gifs and html files but I have no idea who the author is)] [index] (9) Base sequence (a) The sequence of bases in a DNA molecule represent information (b) This sequence is effectively unconstrained by the structure of the double helix (c) As a consequence, much of the DNA in a chromosome (i.e., that which makes up genes) represents unique nucleotide sequences (d) The rest consists of various repeated sequences which typically are species specific (e) [base sequence (Google Search)] [index] (10) Strand complementarity (a) Because of base pairing and the making up of a double helix of DNA of two separate strands, there exists a redundancy of information carried by the double helix (b) Note, however, that the two DNAs do not possess the same sequence (c) Instead, each possesses the complementary sequence of the other (d) Another way of saying this is that through base pairing one strand is capable of specifying the sequence of the other strand, and vice versa (e) This sequence complementarity forms the basis of DNA-templated DNA polymerization (i.e., DNA replication) (f) [strand complementarity (Google Search)] [index] (11) Semiconservative DNA replication (a) The specific mechanism by which DNA is replicated is termed semiconservative

Despite the long, confusing word used to describe it, this is actually the simplest mechanism by which template-dependent DNA replication might occur (c) In short, semiconservative DNA replication consists of each strand of DNA in a double helix specifying the polymerization of a new strand which, in turn, remains attached to its parent strand (d) This parent-daughter strand forms a new double helix that consists of both a parental strand of DNA and a newly synthesized strand of DNA (e) (note that above I am using the term strand synonymous to single molecule of DNA, i.e., half of a double helix) (f) See Figure 16.7, A model for DNA replication: the basic concept (g) [semiconservative DNA replication (Google Search)] [index] (12) 5 3 polarity (a) Recall that the sugars of nucleic acids are numbered with primes (i.e., 1 through 5) (b) Recall additionally that the backbone of polymerized nucleic acids consists of the 3 through 5 carbons alternating with a covalently bonded phosphate group (c) See Figure 16.11, Incorporation of a nucleotide into a DNA strand (d) [5' 3' polarity (Google Search)] [index] (13) Antiparallel strands (a) Recall additionally that the two DNA strands that make up a double helix are arranged antiparallelly (b) That is, starting from one end of the double helix, one strand runs in the 5 3 direction while the other runs in the 3 5 direction (c) See Figure 16.12, The two strands of DNA are antiparallel (d) This antiparallel nature of DNA impacts on DNA replication (e) [antiparallel strands (Google Search)] [index] (14) 5 3 direction of synthesis (a) During DNA synthesis, incoming subunits arrive with phosphates (b) They attach to the 3 OH exposed at the end of the growing new strand (c) This supplies the phosphate making up the sugar-phosphate backbone of DNA (d) It also constrains the growth of the new DNA strand to the 5 to 3 direction (e) That is, for each DNA molecule there exists a 5 end at which no synthesis is occurring (directly, anyway) and a 3 end at which synthesis may occur (f) See Figure 16.11, Incorporation of a nucleotide into a DNA strand (g) [5' 3' direction of synthesis (Google Search)] [index] (15) Nucleosides (a) The incoming subunit, in fact, does not carry just one phosphate (b) Instead it carries three phosphates (i.e., their structure is analogous to that of ATP) (c) Nucleosides that carry a single phosphate are called nucleotides and this is what remains following the addition of a nucleoside triphosphates to a growing DNA (or RNA) polymer

(b)

The hydrolytic removal of two of these phosphates supplies the energy employed to attach the subunit to the 3 OH of the growing DNA strand (e) See Figure 16.11, Incorporation of a nucleotide into a DNA strand (f) [nucleoside -HIV (Google Search)] [index] (16) DNA polymerase (a) The enzyme that catalyzes this template-directed conversion of nucleosides into an elongated DNA strand is called DNA polymerase (b) Note that DNA polymerase can elongate a strand of DNA only in the 5 3 direction (c) See Figure 16.11, Incorporation of a nucleotide into a DNA strand (d) See Figure 16.12, The two strands of DNA are antiparallel (e) [DNA polymerase (Google Search)] [index] (17) RNA priming (a) DNA polymerase attaches new nucleotides with high fidelity (thus reducing errors) (b) This high-fidelity nucleotide addition requires the existence of a 3 OH (c) This means that DNA polymerase cannot initiate DNA replication since, at the start of DNA replication, the to-be-synthesized DNA strand does not yet possess a 3 OH (i.e., the strand does not yet exist) (d) This problem of how to initiate DNA replication in the absence of a 3 OH is solved by priming using RNA (e) See Figure 16.14, Priming DNA synthesis with RNA (f) [RNA priming (Google Search)] [index] (18) Primase (a) DNA replication is initiated with RNA by an enzyme called primase (b) Primase can initiate template-directed polymerization without a 3 OH (c) Thus, DNA polymerase uses a RNA 3 OH to initiate replication (d) The RNA is then eventually replaced by DNA (e) Note that replacing the RNA with DNA at the very ends of linear chromosomes is a problem (no matter what, the very end will never have a 3 OH), thus explaining, in part, the problem of telomere erosion in eukaryotes (f) See Figure 16.14, Priming DNA synthesis with RNA (g) [primase (Google Search)] [index] (19) Origins of replication (a) One place that primase acts is at certain DNA sequences called origins of replication (b) This is the site of priming of the leading strand of DNA replication (c) See Figure 16.10: Origins of replication in eukaryotes (d) [origins of replication (Google Search)] [index] (20) Replication fork (a) One role of these replication origin proteins is to open up the double helix so that both strands are exposed as single-strand DNA, i.e., as potential templates (b) The local bubble created by this separation of strands about the origin is bordered at each end with a replication fork

(d)

It is at these replication forks that the parent double helix is unwound and daughter DNA strands are synthesized, thus converting one double helix into two (d) See Figure 16.10: Origins of replication in eukaryotes (e) [replication fork (Google Search)] [index] (21) Leading strand (a) Note that because of the antiparallel nature of the double helix, as the replication fork opens, for one DNA strand the opening occurs in the 3 5 direction while for the other DNA strand the opening occurs in the 5 3 direction (b) See Figure 16.12, Synthesis of leading and lagging strands during DNA replication (c) Note that both new daughter strands are laid down in a 5 3 direction antiparallel to each template (parent) strand (d) As a consequence, for only one daughter strand will the replication fork be opening such as to allow unimpeded 5 3 synthesis (e) This unimpeded strand is called the leading strand (f) [leading strand (Google Search)] [index] (22) Lagging strand (a) The other strand must be replicated in the direction leading away from the replication fork (b) Consequently, the replication of this other strand is discontinuous (c) Its replication must wait for the replication fork to sufficiently open up the DNA so that a reasonably large number of nucleotides are exposed (on the order of 100 to 1000 depending on system) (d) Then at the replication fork RNA synthesis must be primed, thus priming DNA synthesis which then proceeds in the 5 3 direction (e) At the other end DNA polymerase eventually (a fraction of a second later) bumps into the RNA from a previous priming (f) This RNA is stripped away by the DNA polymerase and replaced with DNA (g) The new segment of DNA is then ligated to the downstream DNA strand (h) This represents the complex synthesis of the lagging strand (i) See Figure 16.12, Synthesis of leading and lagging strands during DNA replication (j) [lagging strand (Google Search)] [index] (23) Okazaki fragments (a) The fragments of DNA synthesized to make up the lagging strand are called Okazaki fragments for their discoverer (b) [okazaki fragment or fragments (Google Search)] [index] (24) DNA ligase (a) The enzyme that ligates together the Okazaki fragments is called DNA ligase (b) [DNA ligase (Google Search)] [index] (25) The replication fork, a summary (a) See Figure 16.16, A summary of DNA replication

(c)

(b)

Note:

(i) The helicase enzyme (ii) Single-stranded binding protein (iii) Proofreading (c) [replication fork (Google Search)] [index] (26) Helicase (a) The enzyme that opens the replication fork is called helicase (b) This name refers to the fact that the double helix is unwound (helically, get it?) at the replication fork (c) [helicase (Google Search)] [index] (27) Single-strand binding protein (a) An unwound double helix is unstable (b) To prevent the individual strand from reannealing prior to the synthesis of the new daughter strand, a protein is employed to stabilize the singlestranded DNA (c) This protein is called single-strand binding protein (d) [single strand binding protein (Google Search)] [index] (28) Proofreading (a) In addition to all of the above (and much not mentioned) another problem run into during DNA replication is that template directed replication is not sufficient to achieve the high fidelity of DNA replication that organisms achieve (b) That is, the interaction between complementary bases is not precise enough to allow the level of DNA replication fidelity most organisms shoot for (c) An additional level of fidelity is achieved by what is known as proofreading (d) During DNA replication, the newly attached bases are checked to make sure they really are the correct, complementary bases (e) Those that are not are removed and replaced (f) In prokaryotes this is yet another function of the DNA polymerase while eukaryotes (in all their complexity) use additional proteins (g) RNA viruses, like HIV and influenza virus, by the way, do not employ proofreading and consequently possess much higher mutation rates than do most DNA-based organisms; this high mutation rate allows HIV (and influenza virus, etc.) to evolve maddeningly quickly (h) Finally, note that your text on page 290 seems to confuse mutation and DNA damage (as in Fortunately, these changes, or mutations, are usually corrected); try not to let this get to you (i) [proofreading replication (Google Search)] [index] (29) Telomeres (telomerase) (a) The end of a linear chromosome presents an additional DNA replication problem: At the end of a chromosome RNA priming cannot supply a 3 OH (b) Why not? There is no sequence beyond the end of the chromosome to template the polymerization of the priming RNA sequence

As a consequence, the ends of linear chromosomes tend to erode with every replication (i.e., the very ends arent replicated so are lost, and this effect is cumulative so that each chromosomal replication results in a loss of additional DNA) (d) To guard against this erosion, eukaryotes possess regions of DNA at the end of their chromosomes called telomeres that serve essentially as DNAerosion buffers (e) That is, the telomeres, which are otherwise not important for chromosome functioning, erode rather important parts (e.g., protein-coding regions) (f) To replace eroded telomeres, eukaryotes employ an enzyme called telomerase (g) Telomerase, however, is mostly found in cells that are immortal (e.g., germ line cells) or in the developing organism (h) The absence of telomerase places an upper limit on how many times the cells in your body may divide, thus providing an additional level of protection against uncontrolled cell growth such as that seen with cancer (i) See Figure 16.19, Telomeres and telomerase (j) [telomere or telomeres, telomerase (Google Search)] [index] (30) (Review transcription if you have time) (31) Vocabulary [index] (a) Antiparallel strands (b) Base sequence (c) Chargaff (d) Chromosomes (e) DNA ligase (f) DNA polymerase (g) Helicase (h) Lagging strand (i) Leading strand (j) Nucleosides (k) Okazaki fragments (l) Origins of replication (m) Primase (n) Proofreading (o) Replication fork (p) RNA priming (q) The replication fork, a summary (r) Semiconservative DNA replication (s) Single-strand binding protein (t) Strand complementarity (u) Telomerase (v) Telomeres (w) 5 3 direction of synthesis (x) 5 3 polarity

(c)

Course-external links are in brackets Click [index] to access site index Click here to access texts website Vocabulary words are found below (1) Chapter title: From Gene to Protein (a) The DNA inherited by an organism leads to specific traits by dictating the synthesis of certain proteins. Proteins are the links between genotype and phenotype. (b) [from gene to protein (Google Search)] [index] (2) Central dogma of molecular genetics (a) The central dogma of molecular genetics is typically depicted as a shorthand review of how genetic information moves around a cell, or from parent to offspring. (b) The central dogma looks like this: (i) DNA DNA RNA protein (c) Note that we can give names to these various steps: (i) DNA DNA = replication (direction of arrow is arbitrary) (ii) DNA RNA = transcription (direction of arrow is not arbitrary) (iii) RNA protein = translation (ditto) (d) This chapter deals particularly with the last two, transcription and translation (e)

(f)

(g)

(reverse transcription serves as an exception to the central dogma as originally conceived; it consists of DNA RNA, i.e., RNA DNA, and is employed by such things as retroviruses including the virus that causes AIDS; note in the above figure that, of course, proteins also serve as enzymes) [central dogma, central dogma molecular genetics (Google Search)] [index]

(3) RNA (uracil) (a) See Figure 17.2, Overview: the roles of transcription and translation in the flow of genetic information (b) RNA is a nucleic acid polymer that resembles DNA except (i) RNA uses the sugar ribose instead of deoxyribose Ribose has an OH group at the 2 carbon instead of the H seen with deoxyribose found in DNA (ii) RNA employs the nitrogenous base uracil (U) instead of the pyrimidine thymine For the latter point, that is, T U The analogous base-pairing is U-A Note that U is energetically cheaper to make than T but that U is also less stable than T (c) [RNA, uracil (Google Search)] [index] (4) One gene-one polypeptide hypothesis (a) Beadle and Tatum developed the one gene-one enzyme hypothesis in the 1940s (b) The idea is that Mendels hereditary units are found in DNA but work by specifying enzymes (c) This hypothesis was modified to one gene-one protein since not all proteins are enzymes but genes work by specifying proteins (d) Finally, this hypothesis was modified to one gene-one polypeptide since many proteins consist of more than one polypeptide (e) Genes specify the construction of specific polypeptides (f) (in fact, to deconstruct things further, genes specify the transcription of specific RNAs) (g) See Figure 17.1, Beadle and Tatums evidence for the one geneone enzyme hypothesis (h) ["one gene one protein", "one gene one polypeptide", "one gene one peptide" (Google Search)] [index]

TRANSCRIPTION
(5) Transcriptionintroduction (template strand) (a) The DNA RNA flow of genetic information is termed transcription (b) The term transcription reflects that the information in DNA (i.e., nucleotide sequence) is copied into a similar code in RNA (c) Only one strand of the two possible strands of DNA is typically copied (always one strand per transcriptional unit) (d) In different places on a chromosome the other strand may be copied (e) The DNA strand that provides the complementary template to RNA polymerization is called the template strand (f) The RNA can be of a number of types including: (i) Messenger RNA (mRNA) (ii) Transfer RNA (tRNA)

(iii) Ribosomal RNA (rRNA) (iv) Etc. (e.g., spliceosomes) (g) See Figure 17.2, Overview: the roles of transcription and translation in the flow of genetic information (h) (see transcription in detail, below) (i) [RNA transcription, template strand (Google Search)] [index] (6) Messenger RNA (mRNA) (a) If the RNA produced by transcription is to be used to code for the synthesis of proteins, it is called messenger RNA (a.k.a., mRNA) (b) [messenger RNA, mRNA (Google Search)] [index] (7) Transfer RNA (1) (tRNA) (a) Another category of RNA, used during protein synthesis to ferry amino acids to growing peptide chains, is called transfer RNA (a.k.a., tRNA) (b) (for more information, see transfer RNA, below) (c) [transfer RNA, tRNA (Google Search)] [index] (8) Ribosomal RNA (rRNA) (a) Another category of RNA that together constitute about 60% of the mass of ribosomes is called ribosomal RNA or rRNA (b) (in Escherichia coli cells, ribosomes make up 25% of the dry weight of cells) (c) [ribosomal RNA, rRNA (Google Search)] [index] (9) Translationintroduction (a) The RNA protein flow of genetic information is termed translation (b) The term translation reflects that the information in mRNAs (i.e., nucleotide sequence) is translated into a new language, i.e., amino acid sequence (c) See Figure 17.2, Overview: the roles of transcription and translation in the flow of genetic information (d) (see translation in detail, below) (e) [protein translation (Google Search)] [index] (10) Eucaryotic segregation of transcription and translation (a) Note that due to the existence of the nuclear membrane in eucaryotes, there exists a temporal and spatial separation of transcription and translation (b) See Figure 17.2, Overview: the roles of transcription and translation in the flow of genetic information (c) Transcription occurs within the nucleus, where the DNA resides (d) Translation occurs within the cytosol, where the functional ribosomes reside (e) There is no such segregation of transcription and translation in prokaryotes (f) [segregation of translation and transcription (Google Search)] [index] (11) Codons (a) The DNA and RNA nucleotide sequence code consists of one of four types of nucleotides (4 each, that is) (b) The amino acid sequence code consists of 20 amino acids

In translating from nucleotide sequence to amino acid sequence there cannot be a one-to-one correspondence (4 < 20) (d) There also cannot be a two-to-one correspondence (42 < 20) (e) Instead there exists a three to one correspondence (43 > 20) (f) The three nucleotides that specify an amino acid during translation are called codons (g) See Figure 17.3, The triplet code (h) See Figure 17.4, The dictionary of the genetic code (i) [codons or codon (Google Search)] [the genetic code (the table of codons and what that means) (Shaun D. Black)] [index] (12) Codons are a property of mRNA (a) Note that codons exist in mRNA, but only their complement exists on the template strand of DNA (b) (though note, additionally, that on the non-template strand of DNA the analogous DNA codonsthough without uracilexists) (c) See Figure 17.4, The dictionary of the genetic code (d) [codons mRNA (Google Search)] [index] (13) Redundancy of triplet code (a) 43 = 64 >> 20 (b) Consequently, there are many more codons than there are amino acids (c) However, 61 of the 64 possible codons do code for an amino acid (d) This is because many amino acids are specified by more than one codon (e) See Figure 17.4, The dictionary of the genetic code (f) (no, you dont have to memorize the figure) (g) [triplet code redundancy OR redundant (Google Search)] [index] (14) Lack of ambiguity in the triplet code (a) Note that while the code is redundant, it is not ambiguous (b) That is, each codon specifies for one and only one amino acid, not more than one (c) [triplet code ambiguity (Google Search)] [index] (15) Codons dont overlap (a) Another property of codons is that they are arrayed one after another in the mRNA (b) That is, they do not overlap (c) (note that there is an only slightly related exception in which codons can overlap and this is when reading frames of different genes overlap) (d) See Figure 17.3, The triplet code (e) [codons overlap (Google Search)] [index] (16) There is no punctuation between codons (a) Furthermore, codons do not have gaps between them (i.e., there is no punctuation) (b) See Figure 17.3, The triplet code (c) [punctuation codons (Google Search)] [index] (17) Start codon (AUG, methionine) (a) The codon AUG codes for the amino acid methionine (b) See Figure 17.4, The dictionary of the genetic code

(c)

AUG also specifies the initiation of translation Thus, all polypeptides initially begin with methionine (Met) Note that as a part of post-translational protein processing the Met amino acid is often clipped off (f) (though I dont expect you to learn all of the codons and their assignments, you should memorize AUG, methionine, and the fact that it serves as the start codon of reading frames) (g) [start codon, methionine (Google Search)] [index] (18) Stop codons (a) Only 61 of the 64 possible codons specify amino acids (b) The other three specify what are known as stop codons (c) (or nonsense codons to distinguish them from the other 61 sense codons) (d) See Figure 17.4, The dictionary of the genetic code (e) Stop codons instruct the ribosome to stop adding amino acids to the growing peptide chain (f) [stop codon (Google Search)] [index] (19) Reading frame (a) The sequence of codons beginning with AUG and ending with a stop codon is called the reading frame (b) Note that the reading frame consists of (x + 1) * 3 nucleotides where x is the number of amino acids found in the resulting polypeptide (prior to post-translational modification) and the additional 1 is a stop codon (c) [reading frame, open reading frame (Google Search)] [index] (20) (nearly) Universal triplet code (a) The language of codons is nearly universal among extant organisms (b) (e.g., AUG specifies Met and is the start codon in all or nearly all living organisms) (c) This near-universality is taken as evidence that all extant organisms share a common ancestor (d) Furthermore, the divergence from this common ancestor must have occurred at a time after the implementation of the triplet code (e) Since the triplet code is somewhat arbitrary, the converse hypothesis, that all organisms somehow independently adopted the same codons for each amino acid, is much less likely (f) As a consequence of the near-universality of the triplet code, genes from one organism may be transferred into unrelated organisms and still express (i.e., be transcribed then translated) (g) [universal triplet code (Google Search)] [index] (21) Transcription in detail (a) Transcription takes place in three steps (i) DNA binding and initiation (ii) Elongation of the RNA strand (iii) Termination of transcription (b) The primary enzyme involved is called RNA polymerase (c) See Figure 17.6, The stages of transcription: initiation, elongation, and termination

(c) (d) (e)

See Figure 17.25, A summary of transcription and translation in a eukaryotic cell (e) [RNA transcription (Google Search)] [index] (22) RNA polymerase (a) RNA polymerase works similarly to DNA polymerase (b) Like DNA polymerase, RNA polymerase employs a DNA template (i.e., the template strand) but, of course, polymerizes RNA (c) Just as with DNA polymerase, RNA polymerase synthesizes in the 5 3 direction (d) [RNA polymerase (Google Search)] [index] (23) Promoter binding (transcription factor) (a) The first step in transcription is DNA binding (b) In prokaryotes this involves the recognition of specific DNA sequences (promoters) by the RNA polymerase (c) See Figure 17.7, The initiation of transcription in a eukaryotic promoter (d) In either case, the promoter is found upstream from the start codon (e) Once bound the RNA polymerase begins transcribing (i.e., polymerizing RNA from a DNA template) (f) In eukaryotes this involves the binding of RNA polymerase to proteins, called transcription factors, that are involved in sequence recognition (g) [promoter binding (Google Search)] [index] (24) Elongation (1) (a) To initiate transcription, the RNA polymerase must separate the DNA strands of the double helix (b) Throughout the elongation of the RNA transcript, the DNA strand is kept open approximately 10 bases (c) Note that a given gene may be transcribed by more than one RNA polymerase simultaneously, with one RNA polymerase following another along on the transcribed DNA (d) See Figure 17.6, The stages of transcription: initiation, elongation, and termination (e) [transcription elongation (Google Search)] [index] (25) Termination of transcription (a) Just as transcription is initiated at certain base sequences, it is similarly terminated at specific base sequences (b) With termination the RNA transcript is released from the RNA polymerase and DNA template strand, and the RNA polymerase from the DNA (c) See Figure 17.6, The stages of transcription: initiation, elongation, and termination (d) [transcription termination (Google Search)] [index] (26) mRNA processing (a) The compartmentalization of the eukaryotic cell results in a separation of transcription and translation, both spatially and temporally (b) Eukaryotic cells take advantage of this compartmentalization to modify RNAs prior to translation

(d)

Modifications include (i) Addition of a 5 cap (ii) Addition of a poly-A tail (iii) Removal of introns (d) See Figure 17.8, RNA processing: addition of 5 cap and poly(A) tail (e) mRNAs are allowed to leave the nucleus only once they have been processed (f) (recall that translation occurs only in the cytosol) (g) [mRNA processing (Google Search)] [index] (27) Introns and exons (a) Most eukaryotic genes do not exist as continuous reading frames (b) Eukaryotic mRNAs, however, do exist as continuous reading frames (c) The conversion of RNAs that do not possess continuous reading frames to ones that do is a form of mRNA processing (d) The intervening sequences that disrupt reading frames in genes and in RNAs prior to their processing are called introns (i.e., intervening sequences) (e) The sequences which are spliced together, upon the removal of introns, to form a continuous reading frame are called exons (i.e., expressed sequences) (f) See Figure 17.9, RNA processing: RNA splicing (g) [introns exons (Google Search)] [index] (28) Spliceosome (a) There exists a nuclear structure involved in intron excision called a spliceosome (b) Note that yet another form of RNA plays a functional role in spliceosomes (c) See Figure 17.10, The roles of snRNPs and splicosomes in mRNA splicing (d) [spliceosome (Google Search)] [index]

(c)

TRANSLATION
(29) Translation in detail (a) Translation is far more complex than transcription, involving many dozens of distinct macromolecular players (b) See Figure 17.12, Translation, the basic concept (c) These players include (i) Transfer RNAs (ii) Ribosomes (iii) Aminoacyl-tRNA-synthetases (iv) mRNA (v) The growing peptide (d) Analogously, transcription employs DNA, RNA polymerase, and a growing RNA transcript (e) Like transcription, translation occurs in three basic steps

(i) Initiation (ii) Elongation (iii) Termination (f) Note that much of translation is powered by the nucleoside triphosphate GTP (which you last saw during the Krebs cycle) rather than ATP (g) Remember that the primary goal of translation is the synthesis of a polypeptide from mRNA-coded information (h) Also, keep in mind that it is probably much easier to understand translation by following the figures in your text than from simply reading the text or these lecture notes (i) See Figure 17.23, A summary of transcription and translation in a eukaryotic cell (j) [translation RNA (Google Search)] [index] (30) Transfer RNA (2) (a) Transfer RNAs are the translating units (b) One side of the tRNA binds to a specific codon found on an mRNA (c) The other side binds to a specific amino acid (d) See Figure 17.12, Translation, the basic concept (e) See Figure 17.13, The structure of transfer RNA (tRNA) (f) [transfer RNA, tRNA (Google Search)] [index] (31) Anticodon (a) The region of the tRNA that binds to the mRNA codon is called the anticodon (b) See Figure 17.12, Translation, the basic concept (c) Note that the anticodon is more or less complementary to the mRNA codon in terms of base-pairing The Genetic Code (supplemental table) U C A G Phenylalanine U Leucine U C Serine STOP A STOP Tryptophan G U Histidine C Proline Arginine A Glutamine G U Asparagine Serine C Threonine A Lysine Arginine G U Aspartic Acid C Alanine Glycine A Glutamic Acid G Tyrosine Cysteine

Leucine

Isoleucine Methionine

Valine

(d) [anticodon (Google Search)] [index] (32) Wobble (a) This complementarity between codon and anticodon is more or less because the third base of a codon tends to be ambiguously bound by the anticodon (b) Note that much of the variation in the sequence of the codons specifying individual amino acids is found in the third base of the codon (c) The anticodon third-base ambiguous binding is why redundant (i.e., synonymous) codons tend to vary at the third base (d) This tendency of anticodons to bind codons varying in their third base is called wobble (see table to right) (e) See Figure 17.4, The dictionary of the genetic code (to see how the third bases in codons tend to have a more-minor role in specifying the amino acid than the first two bases) (f) See Figure 17.13, The structure of transfer RNA (tRNA) (note that it is the 5 base that is read ambiguously due to wobble) (g) [wobble translation (Google Search)] [index] (33) Aminoacyl-tRNA synthetases (a) tRNAs employ their anticodons to bind specific codons found on mRNAs (b) However, tRNAs are not responsible for specifying what amino acid they attach to (c) Instead there exist enzymes that recognize specific tRNAs (often at the anticodons) and attach specific amino acids (d) These enzymes are called aminoacyl-tRNA-synthetases (e) (yes, it is a big word; sound it out as: amino-acyl-tRNA-syn-theh-tase) (f) The cost of amino acid addition is one ATP (g) See Figure 17.14, An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA (h) There exist at least one aminoacyl-tRNA-synthetase for each amino acid (i.e., 20) (i) [aminoacyl-tRNA synthetase (Google Search)] [index] (34) Ribosomes (A site, P site, E site) (a) Ribosomes are the machines within which tRNAs function to read the mRNA code and translate that code into polypeptides (b) Ribosomes consist of one large and one small subunit (both which are complexes of rRNA and many proteins) (c) Ribosomes have three major binding sites, one each for (i) The mRNA (ii) The tRNA attached to the incoming amino acids (the A site) (iii) The tRNA attached to the growing polypeptide (the P site) (d) Ribosomes additionally have an E site from which tRNAs exit the ribosome (e) See Figure 17.15, The anatomy of a functioning ribosome (f) The ribosomes function is to catalyze the peptide bond formation between the polypeptide held at the P site to the incoming amino acid held at the A site

(g) [ribosome (Google Search)] [index] (35) Initiation (a) Initiation of translation involves the binding of the Met-carrying tRNA to the AUG start codon found on the mRNA that in turn is bound to the small subunit of the ribosome (b) See Figure 17.17, The initiation of translation (c) Note that the mRNA is bound to the ribosome by a ribosome-recognition sequence found on the mRNA (d) Note that the tRNA is found at what will be the P site (e) The large ribosomal subunit then binds to the small subunit (f) The above binding occurs at a cost of one GTP (g) [translation elongation (Google Search)] [index] (36) Elongation (2) (a) Elongation in translation is more complex than that of transcription because there are more players (e.g., tRNAs and ribosomes) and because the mRNA is read three nucleotides (one codon) at a time rather than only a single nucleotide (i.e., as in transcription) (b) Charged tRNAs (i.e., ones to which an amino acid is bound) diffuse into the A site and only those that successfully interact with the mRNA codon stay there (this step actually requires energy to performone GTP) (c) A peptide bond is then formed between the incoming amino acid and the peptide held at the P site (this releases the peptide from the P-site located tRNAno additional energy is required from that already stored in the various molecules involved) (d) The mRNA is then translocated one codon forward so that the tRNA that had held only one amino acid in the A site, but now holds the growing polypeptide, is now found in the P site (e) The translocation step requires one GTP (f) See Figure 17.18, The elongation cycle of translation (g) Note that the ribosome moves along the mRNA in the 5 3 direction the mRNA thus moves through the ribosome in the 3 5 direction (i.e., the 5 end leads) (h) [translation elongation (Google Search)] [index] (37) Termination (a) When the codon in the A site is a nonsense (stop) codon, no associated tRNA exists (b) Instead release factors bind to the stop codon in the A site (c) This causes the now-completed peptide to be hydrolyzed off of the P site tRNA (d) In addition, the ribosome releases the mRNA and then separates into two subunits (e) See Figure 17.19, The termination of translation (f) [translation termination (Google Search)] [index] (38) Post-translational polypeptide modification (a) During and after its synthesis, a polypeptide chain begins to coil and fold spontaneously, forming a functional protein of specific conformation: A

three-dimensional molecule with secondary and tertiary structures. A gene determines primary structure, and primary structure determines conformation. (b) Posttranslational modifications of this folding polypeptide, however, can include (i) Covalent attachment of sugars, lipids, phosphate groups, etc. (ii) Removal of one or more leading (amino) end amino acids (e.g., Met) (iii) Cleavage of polypeptide chain (c) [post-translational modification, posttranslational modification (Google Search)] [index] (39) Signal sequences (a) The targeting of proteins occurs during and after translation (b) An amino-terminal amino acid sequence can play a role in protein targeting (c) Such sequences are called signal sequences (d) Signal sequences function like ZIP codes, addressing proteins to certain locations in the cell. (e) See Figure 17.21, The signal mechanisms for targeting proteins to the ER (f) [signal sequence (Google Search)] [index]

MUTATION
(40) Mutation (a) A mutation is a replicable change in nucleotide sequence (b) Contrast this with DNA damage which is a non-replicable alteration in DNA structure (c) Mutations come in a variety of (often overlapping) categories including (i) Point mutations (ii) Silent mutations (iii) Missense mutations (iv) Nonsense mutations (v) Insertions (vi) Deletions (vii) Frameshift mutations (d) [mutation (Google Search)] [index] (41) Mutations are typically detrimental . . . (a) Mutations represent a change in highly evolved information (b) Typically changes to well functioning systems are detrimental, and mutations are no exception (c) (i.e., mutations are the biological equivalent of a violation of the If it aint broke, dont fix it axiom) (d) [mutations detrimental, mutations bad (Google Search)] [index] (42) but Mutations are the only way to change

Despite the typically detrimental nature of mutations, they also represent the only way in which novel, beneficial information is typically introduced into a genetic system (b) (the other way is horizontal transfer, i.e., from different species, and even then mutations still represent the ultimate source of information) (c) Thus, while mutations typically are detrimental, from time to time a mutation actually increases the survival and reproductive potential of an organism (43) Point mutation (a) Mutations come in a number of flavors, the easiest to envisage being the point mutation (b) A point mutation is simply a change of one nucleotide in a base sequence to a different nucleotide (e.g., a change from A to G) (c) See Figure 17.23, The molecular basis of sickle-cell disease: a point mutation (d) Upon replication that change will be duplicated into the complementary strand of one of the daughter chromosomes (e) [point mutation (Google Search)] [index] (44) Silent mutation (a) Point mutations, even those occurring within exons, need not result in changes to amino-acid sequence (b) Why? Recall that third base substitutions in codons frequently will not result in a change in the amino acid coded for (c) [silent mutation (Google Search)] [index] (45) Missense mutation (a) When a point mutation results in a change in amino acid, that mutation is termed a missense mutation (b) See Figure 17.24, Categories and consequences of point mutations (c) Note that missense mutations may or may not have significant impact on protein structure or function (d) If the mutation occurs in a less crucial region of a polypeptide, or results in a change to a functionally similar amino acid, no significant impact may occur (e) If the mutation occurs in the active site or other crucial region of the polypeptide, significant impact may occur (f) Note that typically any impact will be detrimental (g) [missense mutation (Google Search)] [index] (46) Nonsense mutation (a) A nonsense mutation is a point mutation that results in a change from an amino acid-coding codon (a.k.a., a sense codon) to a stop codon (a.k.a., nonsense codon) (b) See Figure 17.24, Categories and consequences of point mutations (c) Note that nonsense mutations truncate polypeptides (i.e., shortens them) (d) Note also that not all sense codons can be converted to a nonsense codon via only a single point mutation (e) [nonsense mutation (Google Search)] [index]

(a)

(47) Insertion (a) An insertion increases the number of nucleotides in a sequence (b) [insertion mutation (Google Search)] [index] (48) Deletion (a) A deletion decreases the number of nucleotides in a sequence (b) [deletion mutation (Google Search)] [index] (49) Frameshift mutation (a) Insertions or deletions of more or less than multiples of three cause the most significant disruption (b) Such changes are termed frameshift mutations because they change the sequence of the entire gene downstream of the mutation (c) (i.e., they shift reading frames) (d) See Figure 17.24, Categories and consequences of point mutations (e) Frequently such changes result in the formation of an in-frame stop codon which serves to truncate the protein (f) [frameshift mutation (Google Search)] [index]

CODA
(50) What is a gene? (a) Mendelian (classical genetical) concept: Discrete unit of inheritance (b) Morgan (chromosome theory) concept: Locus on a chromosome (c) Watson and Crick (DNA structure) concept: sequence of nucleotides (d) Beadle and Tatum I (biochemical) concept: one gene-one enzyme (e) Beadle and Tatum II (biochemical) concept: one gene-one protein (f) Beadle and Tatum III (biochemical) concept: one gene-one polypeptide (g) Modern (transcriptional) concept: one gene-one RNA (h) Abedon (pedagogical) concept: one gene-one exam question! (ha, ha) (51) Vocabulary [index] (a) A site (b) AUG (c) Aminoacyl-tRNA-synthetases (d) Anticodon (e) Central dogma of molecular genetics (f) Codons (g) Codons are a property of mRNA (h) Codons dont overlap (i) Deletion (j) E site (k) Elongation (1) (l) Elongation (2) (m) Eucaryotic segregation of transcription and translation (n) Frameshift mutation (o) Initiation (p) Insertion

(q) Introns and exons (r) Lack of ambiguity in the triplet code (s) Messenger RNA (t) Methionine (u) Missense mutation (v) Mutation (w) mRNA (x) mRNA processing (y) Nonsense mutation (z) One geneone polypeptide hypothesis (aa) P site (bb) Point mutation (cc) Post-translational polypeptide modification (dd) Promoter binding (ee) Reading frame (ff) Redundancy of triplet code (gg) Ribosomal RNA (hh) Ribosomes (ii) RNA (jj) RNA polymerase (kk) rRNA (ll) Signal sequences (mm) Silent mutation (nn) Spliceosome (oo) Start codon (pp) Stop codons (qq) Template strand (rr) Termination (ss) Termination of transcription (tt) There is no punctuation between codons (uu) Transcription factor (vv) Transcription in detail (ww) Transcriptionintroduction (xx) Transfer RNA (1) (yy) Transfer RNA (2) (zz) Translation in detail (aaa) Translationintroduction (bbb) tRNA (ccc) Universal triplet code (ddd) Uracil (eee) What is a gene (fff) Wobble (52) Chapter title: Microbial Models: The Genetics of Viruses and Bacteria (a) [microbial models: the genetics of viruses and bacteria, the genetics of viruses and bacteria (Google Search)] [index] (53) Relevance of microorganisms

Microorganisms are the most important component of environmental health (b) Microorganisms cause diseases (c) Microorganisms can help heal as well as prevent disease (d) Microorganisms have numerous commercial/industrial applications (e) Mitochondria and chloroplasts are microorganisms (f) Microorganisms serve as model systems (g) Microorganisms are extremely abundant (h) Microorganisms are Fun! (i) See Figure 18.1, Comparing the sizes of a virus, a bacterium, and a eukaryotic cell (54) Types of microorganisms (55) (a) Different kinds of microorganisms include (i) Bacteria (ii) Viruses (iii) Fungi (iv) Algae (v) Protozoa (b) This chapter will consider mostly the first two types on this list, bacteria and viruses (c) [types of microorganisms (Google Search)] [index]

(a)

VIRUSES
(56) Virus distinguishing features (a) Viruses are smaller than bacteria (typically, at least) (b) Viruses are obligate intracellular parasites (some bacteria are also) (c) Viruses are structurally simpler than cellular organisms (d) Viruses possess a relative dearth of metabolic machinery (e) Many viruses have unusual genomes (f) There exists a relative dearth of antiviral "antibiotics" (g) Viruses go through an acellular stage (h) [virus distinguishing features (Google Search)] [index] (57) Viral characteristics (a) Viruses tend to vary in terms of their (i) Genome type (ii) Capsids and envelopes (iii) Host range (iv) Life cycles (b) [viral characteristics (Google Search)] [index] (58) Genome types (a) The genomes of viruses are typically much smaller than the genomes of cellular organisms (b) Virus genomes are also not always composed dsDNA

Depending on virus, genomes can be (i) dsDNA (ii) ssDNA (iii) dsRNA (iv) ssRNA (d) Virus genomes can also take on a variety of configurations, depending on the virus including (i) Linear (ii) Circular (iii) Segmented (more than one DNA molecule, each holding a different gene or genes) (iv) Diploid (most viruses are haploid, though) (e) See Table 18.1, Classes of animal viruses, grouped by type of nucleic acid (f) [virus genome types (Google Search)] [index] (59) Capsids and envelopes (a) Defining characteristic of viruses is their protected extracellular state (b) Protection is achieved via a capsid (c) In addition, an envelope may be present, surrounding the capsid (d) See Figure 18.2, viral structure (e) [capsid, enveloped virus (Google Search)] [index] (60) Capsid (capsomers) (a) A capsid is a protein shell that surrounds and protects a virus genome while the virus is in the extracellular state (b) The proteins that make up the capsid are called capsomers (c) Note that capsids can come in a variety of shapes and levels of complexity (i) Helical (ii) Polyhedral (iii) Complex (d) See Figure 18.2, viral structure (e) [capsid, capsomer OR capsomere (Google Search)] [index] (61) Envelope (a) Some viruses are additionally surrounded by an envelope (b) An envelope is a lipid bilayer located external to the capsid (c) Envelopes are derived from host-cell lipid bilayers (d) In addition to host membrane proteins, envelopes contain virus-coded proteins (e) These virus proteins are involved in host-cell attachment and genome uptake (f) In non-enveloped viruses, the capsid proteins are responsible for facilitating host-cell attachment and genome uptake (g) See Figure 18.2, viral structure (h) [enveloped virus (Google Search)] [index] (62) Host range (a) All viruses are limited in the host cells they may successfully infect (b) One term that describes this limit is host range

(c)

Many viruses are limited to only a single host species Other viruses have broader host ranges, being capable of successfully infecting more than one host species (e) Many viruses are additionally limited in the cell types they are able to infect within a host (f) One determinant of the host range of a virus is the "lock-and-key" fit between the virus capsid or envelope proteins and virus receptors, the latter of which typically consist of host-proteins (or carbohydrates) found on the surface of cells (g) [virus host range (Google Search)] [index] (63) Bacteriophage (phage) (a) Note that host range often plays a role in the naming of viruses (b) One category, ones which infect only bacteria, are called bacteriophages (a.k.a., phage or phages for short) (c) [bacteriophage or phage (Google Search)] [bacteriophage ecology group (MicroDude)] [index] (64) Life cycle (a) Viruses have varied life cycles, some of which are very complex (b) A life cycle, in general, is a series of events that an organism goes through from birth through reproduction (c) The simplified virus life cycle consists of (i) Adsorption to a host cell (ii) Uptake of the virus genome into the cell (iii) Transcription of virus genes (iv) Translation of the resulting virus mRNAs (v) Replication of the virus genome (vi) Packaging of the new virus genomes into capsids (vii) Progeny-virus release from the host cell (d) See Figure 18.3, A simplified viral reproductive cycle (e) Life cycles we will consider in more detail include (i) The lytic life cycle (ii) The lysogenic life cycle (iii) The life cycle of an enveloped animal virus (iv) The life cycle of a retroviruses (f) [viurus life cycle, life cycle (Google Search)] [index] (65) Lytic life cycle (a) A lytic life cycle requires the destruction of the host cell before progeny release may occur (b) This host-cell destruction is called lysis (c) See Figure 18.4, The lytic cycle of phage T4 (d) [virus life cycle (Google Search)] [index] (66) Lysogenic life cycle (prophage, provirus, temperate virus) (a) In a lysogenic life cycle virus progeny are neither produced nor released (i) Temperate virus = a virus capable of going through a lysogenic cycle (ii) Prophage = a bacteriophage whose genome has integrated into its host's genome during lysogenic growth

(c) (d)

Provirus = equivalent to prophage but more generally applicable (e.g., to animal viruses) (b) Note that a temperate virus must have some alternative life cycle, one that results in progeny production and release (c) Typically this alternative life cycle is a lytic one (e.g., phage lambda, a.k.a., ) (d) See Figure 18.5, the lysogenic and lytic reproductive cycles of phage , a temperate virus (e) [lysogenic cycle, prophage, provirus, temperate virus (Google Search)] ["Lytic, Lysogenic, Temperate, Chronic, Virulent, Quoi?" (Bacteriophage Ecology Group)] [index] (67) Prophage and disease (a) Prophages can carry bacterial virulence factors (genes that produce toxins, for example) (b) Often these factors are required for the bacteria to cause disease (c) Examples include those bacteria responsible for shigatoxigenic E. coli, diphtheria, botulism, and scarlet fever (d) [prophage and disease (Google Search)] [index] (68) Provirus and disease (a) Certain animal viruses are capable of entering a proviral state (b) This state allows the virus to remain within the host without inducing a host anti-viral immune response (c) Such viruses include the herpesviruses and retroviruses (d) [provirus and disease (Google Search)] [index] (69) Enveloped viruses (a) Enveloped viruses do not go through a lytic cycle (b) Instead they produce and release progeny "chronically", i.e., without necessarily first destroying the host cell (c) Progeny release often occurs simultaneous with envelope acquisition (d) See Figure 18.6, The reproductive cycle of an enveloped virus (e) [enveloped virus (Google Search)] [index] (70) Retroviruses (reverse transcriptase) (a) One type of enveloped animal virus is the retrovirus (b) Retroviruses are named for their RNA genomes which are converted to DNA in the course of the viral intracellular life cycle (c) The enzyme that accomplishes this feat is called reverse transcriptase (d) Interestingly, once converted to DNA, the virus then makes new viral genomes as well as mRNAs simultaneously, as the same molecule (e) An example of a retrovirus is human immunodeficiency virus (HIV) (f) See Figure 18.7, HIV, a retrovirus (g) [retrovirus, reverse transcription, reverse transcriptase (Google Search)] [index]

(iii)

BACTERIA GENETIC VARIATION

(71) Genetic variation (a) Evolutionary adaptation is dependent on genetic variation (b) Genetic variation comes from two sources (and ultimately only the former) (i) Mutation (ii) Sex (c) [genetic variation (Google Search)] [index] (72) Bacteria mutation (a) Bacteria display a higher per-gene mutation rate compared with morecomplex (larger-genomed) organisms (b) In short, per-genome mutation rates are fairly constant across dsDNAgenomed organismal types, and bacteria simply have fewer genes (and thus more mutations per gene per round of replication) (c) Bacteria additionally replicate faster than do more complex organisms; they thus not only have more mutations per gene per round of replication, they also have more rounds of replication (d) Bacteria also take up very little space and require relatively few resources per round of replication (ditto) (e) The bottom line is that a typical bacterial population can generate a whole lot of mutational variation, very quickly (f) [bacteria mutation (Google Search)] [index] (73) Bacteria sex (a) Bacteria do not exist as diploids, do not undergo meiosis, and do not tie together sex with reproduction; consequently what a human might call sex and what a bacteria might call sex are difficult to reconcile as similar processes (b) Nevertheless, sex at its basis involves recombination between DNAs sourced from different parents (c) Bacteria tend to be naturally adept at recombination (as mechanisms of repair of DNA damage); the trick then is how DNA from different parents ends up within a single cell (d) Various mechanisms allow this to occur (i) Transformation (ii) Transduction (iii) Conjugation (e) All three mechanisms have in common that the DNAs transferred from one cell to another tend to be transferred only as small "snippets" of DNA, rather than whole chromosomes (f) [bacteria sex (Google Search)] [index] (74) Transformation (a) We already considered transformation in terms of Griffith's experiments with Streptococcus in mice (b) Transformation is the uptake of DNA directly from the environment by a bacterial cell (c) Some bacteria are better at this than are others (d) Bacteria that are not good at this (e.g., E. coli) can be induced to pick up DNA as a laboratory artifact

(e) Such induction is important to bioengineering (f) [transformation DNA (Google Search)] [index] (75) Transduction (a) Transduction involves the movement of snippets of DNA from one cell to another as an accidental stowaway within a bacteriophage (b) Some bacteriophages are better at transducing than others (c) Transduction can be distinguished into two types (i) Specialized transduction (ii) Generalized transduction (d) [DNA transduction (Google Search)] [index] (76) Specialized transduction (a) Specialized transduction involves temperate phages (b) Here when the temperate phages excise themselves from their host's genome they sometimes excise adjacent sections of their host's genome (c) This is called specialized because there is a strong bias toward the movement of specific pieces of DNA (i.e., those adjacent to the normal prophage insertion point) (d) See Figure 18.13, Transduction (e) [specialized transduction (Google Search)] [index] (77) Generalized transduction (a) Generalized transduction involves the packaging of host DNA independent of phage DNA (b) Viruses thus-constructed are not capable of infecting new cells (i.e., completing their life cycle) because they lack phage genes (c) However, they are able to carry snippets of host DNA from one bacteria to another (d) See Figure 18.13, Transduction (e) [generalized transduction (Google Search)] [index] (78) Conjugation (a) In conjugation bacteria dock together and purposefully pass DNA, usually from one (called the male) to a recipient (called female) (b) Typically it is plasmids that are passed rather than chromosomal DNA (c) See Figure 18.14, Bacterial mating (d) See Figure 18.15, Conjugation and recombination in E. coli (e) [bacteria conjugation (Google Search)] [index] (79) Plasmids (a) Some bacterial genes are not found on the bacterial chromosome (b) Instead, genes may be located on smaller pieces of DNA called plasmids (c) Plasmids typically do not contain genes essential to host functioning (d) This is because plasmids may be accidentally lost from cells, along with the genes they hold and any associated functions (e) [plasmid OR plasmids (Google Search)] [index] (80) R plasmids (a) Plasmids can hold genes that are useful under certain circumstances (b) One such circumstance is in the face of exposure to antibiotics (c) Plasmids holding anti-antibiotic genes are termed R plasmids

(d) (e) (f) (g)

Bacteria can acquire R (and other) plasmids fully formed via conjugation or transformation Thus, resistance to antibiotics (etc.) need not develop de novo in a bacterial lineage by mutation, but instead may be acquired fully formed (i.e., fully evolved) As a consequence, bacteria are able to acquire resistance to antibiotics much more easily and rapidly than they might were they limited solely to chromosomal evolution [R plasmids (Google Search)] [index]

BACERIA PHENOTYPIC VARIATION (ADAPTATION)

(81) Adaptation (a) The term adaptation has at least two biological meanings (i) Genetic change resulting in greater evolutionary fitness (evolution) (ii) Physiological change resulting in more appropriate interaction with the environment (b) In both cases what is occurring is some kind of organismal (population or individual) change that occurs in response to environmental input (c) [physiological adaptation bacteria (Google Search)] [index] (82) Molecular genetics (a) In this section we will first consider mechanisms of physiological adaptation in bacteria (b) We will then consider mechanisms that input genetic variation into bacterial populations (genetic variation being the first step toward any evolutionary change) (c) In all cases, what we will be considering is various aspects of the field of molecular genetics (d) [molecular genetics (Google Search)] [index] (83) Why adapt? (a) Physiological adaptation is useful because it allows an organism to fine tune its use of resources to better fit its environment (b) The idea is to avoid using cell resources to make products that are readily available from the environment or otherwise not currently needed (c) It makes energetic sense to make or use proteins responsible for certain metabolic processes only when those processes are needed (d) See Figure 18.19, Regulation of a metabolic pathway (e) Note that you have already leaned about one form of adaptation: feedback inhibition (f) Here we will consider control of gene function (84) Operon model (a) Within bacteria many biochemical pathways are catalyzed by a series of enzymes (b) These enzymes in turn are coded by genes

As a matter of both utility and control of gene expression, it is fairly common (in bacteria) for groups of genes responsible for the expression of a biochemical pathway to be simultaneously transcribed as a single mRNA (d) The DNA responsible for the transcription of this mRNA together with certain transcriptional-control sequences is termed an operon (e) [operon model (Google Search)] [index] (85) Operon (cofactor) (a) An operon typically consists of the following components (i) One or more structural genes (ii) A promoter (iii) An operator (b) Additionally, there may be one or more regulatory genes and associated proteins (c) Finally, there typically exist various smaller molecules that serve as cofactors in gene regulation (d) The basic idea is to simultaneously turn on or turn off the expression of a subset of metabolically related enzymes (e) [operon, operon cofactor (Google Search)] [index] (86) Structural gene (a) Genes within operons that produce proteins are called structural genes (b) These are the genes whose expression is controlled by the operator (c) [structural gene (Google Search)] [index] (87) Promoter (a) The promoter is the site of RNA polymerase binding (b) The promoter is found upstream of the first structural gene in the polytranscript (c) [promoter transcription (Google Search)] [index] (88) Operator (a) The operator is a DNA sequence upstream of the first structural gene (b) Protein binding to the operator controls RNA polymerase activity (c) [operator DNA (Google Search)] [index] (89) Regulatory gene (a) The regulatory gene, itself, is not part of the operon proper (b) A regulatory gene produces a protein that binds to the operator (c) When bound, this protein may facilitate operon expression (positive control) (d) Alternatively, the protein, when bound, may inhibit protein expression (negative control) (e) [regulatory gene (Google Search)] [index] (90) Repressor (a) A protein that binds the operator, thus inhibiting operon expression, is termed a repressor (b) [repressor (Google Search)] [index] (91) Corepressor (a) A cofactor that activates a repressor is called a corepressor

(c)

(b)

(c) (92) Inducer (a) An inducer is a cofactor that inactivates a repressor (b) That is, in this case the repressor is active (will inhibit operon expression) until the cofactor is present (after which the repressor no longer inhibits operon expression) (93) Reversible interactions (a) It is important to keep in mind while discussing operons that the various bindings that occur all do so reversibly (b) Specifically, repressors reversibly interact with operators (c) And corepressors reversibly interact with repressors (d) As a consequence, low concentrations of active repressor results in a relative dearth of binding to operators (and thus of operon expression) (e) Similarly, low concentrations of corepressor results in a relative dearth of cofactor-repressor binding and therefore little repressor activation (f) [reversible operon (Google Search)] [index] (94) Trp operon (corepressed operon) (a) The trp operon is an example of a corepressed operon (b) Corepression is a common means of controlling the synthesis of anabolic pathways (c) The basic idea is that when tryptophan concentrations within a cell are adequate, the cell stops making the enzymes required for tryptophan synthesis (d) Here tryptophan within the cell serves as the corepressor (e) That is, excess trp binds the appropriate repressor which in turn shuts off the trp operon (f) See Figure 18.20, The trp operon: regulated synthesis of repressible enzymes (g) [inducible operon (Google Search)] [index] (95) Lac operon (inducible operon) (a) The lac operon is an example of an inducible operon (b) Induction is a common means of controlling the synthesis of catabolic pathways (c) In this case, when no lactose is available in a cell's environment, the cell avoids making the large quantities of the enzymes necessary to digest lactose (d) However, when lactose is present in reasonable quantities, a lactose derivative serves as an inducer (e) That is, the binding of lactose (actually, its derivative) to the lac operon repressor inactivates the repressor, thus allowing expression of the lac operon genes (f) See Figure 18.21, The lac operon: regulated synthesis of inducible enzymes (g) [lac operon (Google Search)] [index]

That is, in this case the repressor is inactive (won't inhibit operon expression) until the cofactor is present [corepressor (Google Search)] [index]

(96) Negative control (a) The lac and trp operons are two examples of negative control of gene expression (b) That is, when the operator is bound, transcription is inhibited (c) [negative control, negative control transcription (Google Search)] [index] (97) Positive control (a) Positive control, in contrast, involves protein-DNA binding that enhances promoter activity (rather than blocking RNA polymerase activity) (b) That is, with positive control, protein binding results in more gene expression from the operon (c) [positive control, positive control transcription (Google Search)] [index] (98) CRP (cAMP receptor protein) (a) CRP stands for cAMP Receptor Protein (b) When CRP binds cyclic AMP (a derivative of ATP), CRP is activated (c) When activated, CRP can bind to a promoter and positively modulate operon activity (d) Cyclic AMP (cAMP) is a signal produced, here, when intracellular glucose concentrations are low (e) The idea is that a cell will preferentially employ glucose as a carbon and energy source (f) When glucose concentrations are high, cAMP is depleted, CRP is not active, and CRP-controlled operons (e.g., lac operon) display reduced expression (g) When glucose concentrations are depleted, cAMP is produced, CRP is activated, and CRP-controlled operons display enhanced expression (h) See Figure 18.22, Positive control: cAMP receptor protein (i) [cAMP receptor protein, catabolite activator protein (Google Search)] [index] (99) Vocabulary [index] (a) Adaptation (b) Bacteria mutation (c) Bacteria sex (d) Bacteriophage (e) CRP (f) Capsid (g) Capsids and envelopes (h) Capsomers (i) cAMP receptor protein (j) Cofactor (k) Conjugation (l) Corepressed operon (m) Corepressor (n) Envelope (o) Enveloped viruses (p) Generalized transduction (q) Genetic variation

(r) Genome types (s) Host range (t) Inducer (u) Inducible operon (v) Lac operon (w) Life cycle (x) Lysogenic life cycle (y) Lytic life cycle (z) Molecular genetics (aa) Negative control (bb) Operator (cc) Operon (dd) Operon model (ee) Phage (ff) Plasmids (gg) Positive control (hh) Promoter (ii) Prophage (jj) Prophage and disease (kk) Provirus (ll) Provirus and disease (mm) R plasmids (nn) Regulatory gene (oo) Relevance of microorganisms (pp) Repressor (qq) Retroviruses (rr) Reverse transcriptase (ss) Reversible interactions (tt) Specialized transduction (uu) Structural gene (vv) Temperate virus (ww) Transduction (xx) Transformation (yy) Trp operon (zz) Types of microorganisms (aaa) Viral characteristics (bbb) Virus distinguishing features (ccc) Why adapt? (100) Chapter title: Genome Organization and Expression in Eukaryotes (a) [genome organization and expression in eukaryotes (Google Search)] [index] (101) Structure of DNA (a) DNA in eucaryotic cells is organized into hierarchical structures (b) The less-condensed structures we've been calling chromatin (c) The most condensed structures we've been calling a chromosome

Note that gene transcription tends to decline as organization/condensation increases; the more organized the DNA (regionally), the less it is expressed (e) [structure of DNA (Google Search)] [index] (102) Chromatin (a) During interphase, DNA that is available for transcription is organized as chromatin (b) Chromatin consists of DNA wound around proteins specialized for the task called histones (c) Higher levels of organization share the descriptive term, chromatin (d) See Figure 19.1a, Levels of chromatin packing (e) [chromatin (Google Search)] [index] (103) Histones (nucleosome) (a) Histones are highly evolutionarily conserved proteins (b) They contain a high proportion of positively charged (basic) amino acids (lysine and arginine) (c) These positive charges interact with the negative charge of the sugarphosphate backbone of DNA (d) Individual histone-DNA complexes are called nucleosomes (e) [histone or histones, nucleosome (Google Search)] [index] (104) Euchromatin (a) Interphase DNA that is arrayed as chromatin or slightly higher levels of organization (e.g., 30-nm fiber) is called euchromatin (b) See Figure 19.1a & 19.1b, Levels of chromatin packing (c) Euchromatin is potentially available for transcription (d) [euchromatin (Google Search)] [index] (105) Heterochromatin (a) More tightly packed (condensed) interphase DNA is called heterochromatin (b) See Figure 19.1.c and 19.1d, Levels of chromatin packing (c) Heterochromatin is less available for transcription compared with euchromatin (d) Barr bodies, i.e., inactive X chromosomes, consist mostly of heterochromatin (e) [heterochromatin (Google Search)] [index] (106) Chromosomes (a) The ultimate level of chromosome condensation is achieved during prophase/prometaphase (b) See Figure 19.1d, Levels of chromatin packing (c) The DNA within chromosomes is generally unavailable for transcription (d) [chromosomes (Google Search)] [index] (107) Plasticity of phenotypes (a) The idea that organisms may adapt physiologically is one aspect of phenotypic plasticity (b) In general, organisms are able to modify their phenotype in response to external cues by varying what genes they express

(d)

We have already considered the control of gene expression in prokaryotes Here we consider the control of gene expression in the generally morecomplex eukaryotes (e) [plasticity of phenotypes, phenotype plasticity (Google Search)] [index] (108) Control of gene expression in eukaryotes, overview (a) Different cells, different genes expressed (i) The various cell types of a multicellular organisms express different genes (b) DNA structure impacts gene expression (i) The physical organization of chromatin makes certain genes available for expression and other genes unavailable (c) Many levels of control of gene expression (i) For genes that are available for expression, regulatory opportunities exist at each step in the pathway from gene to functional protein (d) Transcriptional control is primary means (i) Control of transcription is especially important in determining which genes are expressed (ii) In eukaryotes, the selective binding of transcription factors to enhancer sequences in DNA stimulates transcription of specific genes (e) Control responds to internal and external cues (i) The regulatory activity of some of these DNA-binding proteins is sensitive to certain hormones and other chemical signals (f) See Figure 19.7, Opportunities for the control of gene expression in eukaryotic cells (g) [control of gene expression in eukaryotes (Google Search)] [index] (109) Availability to RNA polymerase (a) In general, the less condensed the DNA, the more potentially available it is to RNA polymerase (b) The more available DNA is to RNA polymerase, the more available it is to transcription (c) [transcription RNA polymerase availability OR access OR accessibility (Google Search)] [index] (110) Methylation (a) Recall that a methyl group is -CH3 (b) Methylation of DNA typically consists of a methylation of the cytosine nitrogenous base (c) In general, the more methylated a strand of DNA (i.e., the more cytosines that are methylated), the less transcriptionally active the DNA (d) Methylation thus represents a second, not necessarily independent modification of DNA (i.e., in addition to condensation) that impacts on transcriptional availability (e) [methylation gene expression (Google Search)] [index] (111) Transcriptional control of gene expression (a) The physical and chemical structure of eucaryotic DNA impacts on gene expression as outlined above

(c) (d)

Additional controls of transcription resemble, though do not duplicate the prokaryotic operon model of control of gene expression (c) Precisely, gene expression is controlled by protein binding to specific regions of DNA (d) Given structural availability, what genes a eukaryotic cell expresses typically is controlled, more often than not, by the specific binding of specific regulatory proteins to specific regions of DNA (e) [transcriptional control of gene expression (Google Search)] [index] (112) Eucaryotic gene anatomy (a) In addition to introns, a major difference between eukaryotic and prokaryotic genes is the existence of sequences called enhancers (b) See Figure 19.8, A eukaryotic gene and its transcript (c) [gene anatomy eukaryote or eukaryotic (Google Search)] [index] (113) Enhancers (a) Enhancers are gene-expression control sequences analogous to geneexpression control sequences found in prokaryotes (b) However, enhancer sequences may be found thousands of bases away from the reading frame (c) The great distance between reading frame and enhancer sequences as well as the distance between enhancers suggests that enhancer sequences are involved with changes of DNA structure that serve to enhance transcription (d) Particularly, proteins (transcription factors) bind enhancer sequences thus increasing the transcriptional availability of a gene (e) See Figure 19.9, A model for enhancer action (f) [transcription enhancers (Google Search)] [index] (114) Transcription factors (a) Transcription factors are proteins that affect transcription by binding to DNA to facilitate RNA polymerase binding (b) Other transcription factors also bind directly to RNA polymerase, affecting what promoter sequences the RNA polymerase will bind (c) By varying the transcription factors synthesized, a cell can vary what array of genes are expressed (d) In this way cells with metabolically related genes found on many different chromosomes may be simultaneously transcribed (e) I.e., similarly expressed genes would have similar promoters and enhancer sequences and thus respond similarly to specific arrays of transcription factors (f) See Figure 19.10, Three of the major types of DNA-binding domains in transcription factors (g) [transcription factors (Google Search)] [index] (115) Environmental influence (a) What decides what transcription factors are synthesized? (b) Generally it is internal or environmental influences (c) Basically, the cell senses changes in the internal or external environment and synthesizes or frees up transcription factors in response

(b)

One kind of environmental influence is cell-to-cell chemical signals called hormones (e) [environmental influence on transcription (Google Search)] [index] (116) Post-transcriptional control (a) While transcription is the level at which eukaryotic gene expression is typically controlled, transcription is not the only way that gene expression may be controlled (b) Recall that a gene is not expressed, technically, until its product is active (c) In the case of genes that code for proteins, this means an active protein product (d) Note that, additionally, how long a gene function is expressed depends on how long the various aspects of expression (mRNAs, proteins) exist prior to their degradation (e) Any mechanism of control of gene expression that acts after the translation of a polypeptide may be termed post-translational control (f) See Figure 19.7, Opportunities for the control of gene expression in eukaryotic cells (g) [post-transcriptional control (Google Search)] [index] (117) mRNA degradation (a) One way in which the extent of expression of a gene may be controlled is via mRNA degradation (b) The faster mRNAs are destroyed in the cytoplasm, the more temporally linked are transcription and translation (c) The more temporally linked transcription and translation, the more rapidly a cell can respond to its environment via transcription (d) This strong temporal linkage between transcription and translation is how prokaryotes achieve rapid adaptation to environmental cues (e) Alternatively, the longer mRNAs last, the more protein synthesis which may be acquired per mRNA produced, thus reducing some of the cost of protein synthesis (f) [mRNA degradation (Google Search)] [index] (118) mRNA activation/inactivation (a) mRNAs in eukaryotic cells, in addition to posttranscriptional modification, typically require activation via specific protein binding in order for subsequent translation to take place (b) This protein binding is another step at which control of gene expression may occur (c) For example, whole arrays of mRNAs may be synthesized but not expressed until a time that is appropriate, such as following the fertilization of an egg (d) [mRNA activation, mRNA inactivation (Google Search)] [index] (119) Protein degradation (a) Just as with mRNA degradation, a cell may respond to its environment more quickly by selectively degrading cellular proteins (b) Typically degradation involves the recycling of damaged proteins into constituent amino acids

(d)

By assuring that proteins are turned over with time, a cell is able to change its phenotype to better match environmental conditions (d) See Figure 19.12, Degradation of a protein by a protesome (e) [protein degradation (Google Search)] [index] (120) Protein activation/inactivation (a) A less permanent, and more rapid means of changing phenotype via a modification of protein expression is the simple activation of and inactivation of cellular proteins (b) In this way a cell can avoid wasting proteins (i.e., destroying proteins that may soon be needed) while simultaneously rapidly adapting to environmental cues (c) It is via protein activation and inactivation that eukaryotic cells achieve their more-rapid cellular adaptation to environmental conditions (d) [protein activation, protein inactivation (Google Search)] [index] (121) Vocabulary [index] (a) Availability to RNA polymerase (b) Chromatin (c) Chromosomes (d) Control of gene expression in eukaryotes (e) Enhancers (f) Environmental influence (g) Eucaryotic gene anatomy (h) Euchromatin (i) Heterochromatin (j) Histones (k) Methylation (l) mRNA activation/inactivation (m) mRNA degradation (n) Nucleosome (o) Plasticity of phenotypes (p) Post-transcriptional control (q) Protein activation/inactivation (r) Protein degradation (s) Structure of DNA (t) Transcription factors (u) Transcriptional control of gene expression (122) Chapter title: DNA Technology (a) DNA technology is the chemical manipulation of the genotypes and resulting phenotypes of organisms such that living organisms are modified; alternatively, no-longer-living organisms or their no-longer-living parts may be analyzed chemically at the level of genotype (b) The use of DNA technology has revolutionized how scientists study the genetics,

(c)

biochemistry, even the ecology and evolutionary biology of organisms, plus has allowed the development of novel biological products, indeed whole industries are now devoted to DNA-technology-based production and analysis of biological materials (c) [DNA technology (Google Search)] [index] (123) Genetic engineering (a) Genetic engineering is the artificial manipulation of the genetic material of organisms, including the creation of novel genetic material (i.e., novel nucleotide sequences) (b) This manipulation occurs to a large extent external to organisms, e.g., in test tubes, a.k.a., in vitro (meaning, literally, in glass) (c) Genetic engineering is employed to (i) Make recombinant DNA (ii) To purposefully change nucleotide sequences (iii) To clone DNA (d) In short, genetic engineering represents the manipulation of an organisms genotype by artificial, typically very direct means (e) [genetic engineering (Google Search) [index] (124) Biotechnology (a) Contrasting with genetic engineering, biotechnology refers to the engineering of phenotype (b) Biotechnology is not limited to the manipulation of phenotype by directly manipulating genotype (i.e., via genetic engineering) though today this is very often how phenotypes are manipulated (often to the detriment of more traditional means such as plant and animal breeding) (c) [biotechnology (Google Search)] [index] (125) Molecular techniques (a) Together the manipulation and analysis associated with DNA technology, genetic engineering, and biotechnology are based on a number of technologies generally referred to as molecular techniques (b) Molecular techniques include (though are not limited to) (i) Gene cloning (which is associated with various molecular techniques including in vitro restriction enzyme digests, and DNA ligation plus additional, less-artificial manipulations including transformation and transduction) (ii) Creation of cDNA (iii) Polymerase chain reaction (iv) Gel electrophoresis (v) Various blotting techniques (Southern blotting, Northern blotting, Western blotting, etc.) (vi) RFLP analysis (vii) DNA sequencing (c) [molecular techniques (Google Search)] [index] (126) Molecular biology (a) What is molecular biology? Good question

Basically molecular biology consists of all of the above: DNA technology, genetic engineering, biotechnology, and molecular techniques (c) Some consider molecular biology to be a science; others consider molecular biology to be a collection of techniques (e.g., as listed above) (d) Sometimes people use the phrase molecular biology when they really ought to be using the phrase molecular genetics (e) Basically, this chapter represents an introduction to molecular biology (while chapters 16, 17, 18, and 19 dealt with various aspects of molecular genetics) (f) At least for the past 20 years or so (perhaps and then some) molecular biology has been a hot ticket toward earning money as a biological researcher, and more and more disciplines are jumping on the molecular biology bandwagon, thereby making an understanding of molecular biology and molecular techniques almost (but not quite universally) a prerequisite to employability as a modern biological researcher; translation: if you want to do biology and not do molecular biology, then youve got to be very good at (and very dedicated to) whatever it is that you otherwise do (update, 3/12/02: even Ive started using molecular techniques in my research) (g) [molecular biology (Google Search)] [index] (127) Gene cloning (a) Gene cloning consists of a number of molecular techniques that ultimately serve to place a defined segment of DNA within an organism, typically a different organism from which the DNA was originally derived, such that the DNA segment may be replicated repeatedly within the recipient organism (b) To understand gene cloning, it is becoming traditional to walk students through the steps involved in a typical application of gene cloning (c) See Figure 20.1, An overview of the how bacterial plasmids are used to clone genes for biotechnology (d) Steps involved in gene cloning include: (i) Isolating DNA from the cell of an organism (including digestion with restriction enzymes) (ii) Insertion of that DNA into a plasmid (iii) Placement of the plasmid into a second cell (iv) Measures taken to make sure that the cloned DNA is the DNA of interest (v) Various manipulations of the DNA including subcloning, sequencing, and expression (e) For cloning genes or other pieces of DNA, plasmids are first isolated from bacterial cells. FIGURE 20.1 follows one plasmid as a foreign gene from a eukaryotic cell, in this exampleis inserted into it. The plasmid is now a recombinant DNA molecule combining DNA from two sources. The plasmid is returned to a bacterial cell, which then reproduces to form a cell clone. The foreign gene carried by the plasmid is cloned at the same time, for the dividing bacterium continues to replicate the recombinant

(b)

plasmid. Under suitable conditions the bacterial clone will make the protein encoded by the foreign gene. (f) [gene cloning, cloning DNA (Google)] [index] (128) Restriction enzymes (a) The actual isolation of DNA is fairly straightforward involving the breaking open of cells and subsequent purification of the DNA component [isolation of DNA links (MicroDude)] (b) The actual molecular manipulation of DNA begins only once the DNA is purified, and involves to a large extent the cutting of DNA at specific nucleotide sequences by proteins known as restriction enzymes (c) [restriction enzyme, restriction endonuclease (Google Search)] [index] (129) Restriction site (restriction fragment) (a) The actual nucleotide sequence on a piece of DNA that a restriction enzyme cuts is called a restriction site (b) Most restriction sites are palindromes with identical sequences regardless of the direction one moves down the DNA (keeping in mind, of course, that DNA is antiparallel such that one moves down or up a different strand if one switches direction; example of a palindromes found in the English language, sort of: Nodeba Bob Abedonmy alter ego; see also: Leos Palindrome Collection and Worlds First Palindromic URL?) (c) Restriction enzymes cut covalent phosphodiester bonds of both strands, often in a staggered way, as indicated in the diagram. Since the target sequence usually occurs (by chance) many times in a long DNA molecule, an enzyme will make many cuts. Copies of a DNA molecule always yield the same set of restriction fragments when exposed to that enzyme. In other words, a restriction enzyme cuts a DNA molecule in a reproducible way. (d) See Figure 20.2, Using a restriction enzyme and DNA ligase to make recombinant DNA (e) [restriction site, restriction fragment (Google Search)] [index] (130) Sticky ends (a) Note that most restriction enzymes do not make blunt cuts (b) That is, upon restriction digestion, DNA will contain short single-stranded regions at their ends (c) See Figure 20.2, Using a restriction enzyme and DNA ligase to make recombinant DNA (d) These short regions are termed sticky ends because two DNAs cut by the same restriction enzyme (or even different enzymes if they produce overhangs of the same sequence) can hydrogen bond together via nitrogenous base pairing (e) Thus, a double-stranded DNA (double helix) even though it has been digested by restriction enzymes is still to some extent capable of holding together as a cut-but-still-intact double helix

However, note that these hydrogen-bonded fragments are not strongly bonded together, i.e., following restriction digestion the tendency is for restriction fragments to separate and then to only transiently reattach (and if more than one complementary sticky end is present, reattachment is not necessarily in the same order fragments were in within the original DNA molecule) (g) [sticky ends (Google Search)] [index] (131) DNA ligase (a) Sticky ends (as well as blunt ends) may be covalently bonded together using the enzyme DNA ligase; see figure to right (b) See Figure 20.2, Using a restriction enzyme and DNA ligase to make recombinant DNA (c) Recall that DNA ligase is normally employed by cells during DNA replication (as well as during the repair of DNA damage) (d) [DNA ligase (Google Search)] [index] (132) Recombinant DNA (a) Once the DNA coming from two different organisms has been ligated together, we can now call it recombinant DNA (b) [recombinant DNA (Google Search)] [index] (133) Cloning vector (a) Ligating restriction fragments together in a useful way and order is as much a science as it is an art (heck, its engineering: genetic engineering) (b) When cloning DNA a typical step that follows DNA isolation and restriction digest is the insertion of that DNA (i.e., ligation) into a cloning vector (c) Cloning vectors are typically plasmids and may also be the partial genomes of phages (d) The idea behind a cloning vector is that it is a piece of DNA whose job it is to carry pieces of DNA isolated from one organism into another organism where that DNA (along with the vector in which it is contained) is then replicated and the piece of DNA from the first organism may be expressed (e) See Figure 20.3, Cloning a human gene in a bacterial plasmid: a closer look (f) Note that the vector is also subject to restriction digestion before the insertion of foreign DNA; this creates an insertion point that, ideally, possesses the same or similar sticky ends to those possessed by the

(f)

restriction-digested foreign DNAthus the foreign DNA may hydrogen bond into the cloning vector and then may be bonded covalently following incubation in the presence of DNA ligase (g) Note that cloning vectors can be quite sophisticated, possessing various means by which bacteria that contain the vector may be positively selected from bacteria that do not (e.g., via the use of antibiotic resistance genes) as well as various means by which the presence of an inserted fragment may be visualized (e.g., by the disruption of a copy of the lacZ gene, which codes for -galactosidase, the enzyme that allows Escherichia coli to digest the sugar lactose) (h) Cloning vectors are taken up by cells by transformation or by transduction (i) [cloning vector (Google Search)] [index] (134) Identification of the proper clone (a) One of the most difficult steps in gene cloning is making sure that one has succeeded in cloning the desired DNA (b) First, one has to make sure that the organism being cloned has successfully taken up the cloning vector (e.g., via the presence of antibiotic resistance genes in cloning vectors and antibiotics in growth mediaall bacteria notpossessing the cloning vector will be killed by the antibiotic, thereby only bacteria that possess the cloning vector will live and produce bacterial colonies) (c) Second, one has to make sure that the cloned vector contains an insertion of DNA (e.g., via phenotypic visualization of the disruption of the lacZ gene) (d) Third, one has to confirm either phenotypically or genotypically that the inserted DNA is indeed the DNA that one is interested in (e) Phenotypic identification can involve looking for a specific enzyme activity or via the presence of a new gene product within a cell of an appropriate size and/or confirmed by immunological (antibody) reactivity (f) Genotypic identification can involve various nucleic-acid probing techniques or the actual sequencing of DNA (g) See Figure 20.4, Using a nucleic acid probe to identify a cloned gene (135) Subcloning (a) For whatever it is worth, even when one has successfully cloned a piece of foreign DNA that one is interested in, that is often only a first step of genetic manipulation (b) Often one additionally subclones the foreign gene into more-specialized cloning vectors in order to additionally (e.g., experimentally) manipulate the gene or its products; often, though, this subcloning is easier than the original cloning and identification steps (c) [subcloning (Google Search)] [index] (136) Expression vector (a) One type of specialized cloning vector is an expression vector, a cloning vector designed specifically to express genes at high levels, under strong experimental control, or both (b) [expression vector (Google Search)] [index]

(137) cDNA (complementary DNA) (a) Because of the presence of introns in eukaryotic genes, the expression of eukaryotic genes cloned into bacteria can be problematic even if the eukaryotic DNA control sequences are replaced by bacterial ones (e.g., by cloning into an expression vector) (b) A common way around this is to clone eukaryotic genes from cDNA rather than from genomic DNA (c) cDNA is synthesized from a mature mRNA template (i.e., fully processed with introns removed) using reverse transcriptase enzyme (d) See Figure 20.5, Making complementary DNA (cDNA) for a eukaryotic gene (e) A reason to use eukaryotic host cells for expressing a cloned eukaryotic gene is that many eukaryotic proteins are heavily modified after translation, often by the addition of lipid or carbohydrate groups. Bacterial cells cannot perform any of these processing functions, and if the gene product requiring such processing is from a mammal, even yeast cells will not be able to modify the protein correctly. The use of host cells from an animal or plant cell culture may therefore be necessary. (f) [cDNA, complementary DNA (Google Search)] [index] (138) Polymerase chain reaction (PCR) (a) An alternative means of generating large numbers of copies of a specific piece of DNA (i.e., other than by cloning DNA) is to employ polymerase chain reaction (PCR) (b) In polymerase chain reaction, one employs short DNA primers that are complementary to the opposite ends of a specific sequence of DNA one in interested in amplifying in number (again, keep in mind that DNA is antiparallel and that consequently opposite ends means also complementary to opposite strands) (c) The primers supply the 3 OH primer necessary for the initiation of DNA replication (d) Polymerization is used to produce double-stranded DNA (i.e., a double helix) using single-stranded DNA as the template (e) Individual single-strands of DNA are typically formed via the unwinding of a DNA double helix by the application of heat (f) Thus, PCR consists of heat treatment to unwind DNA, binding of primers to the resulting single-stranded DNA, polymerization of new DNA to form a new double-stranded DNA double helix, repeat

See Methods: The Polymerase Chain Reaction (PCR) found on page 371 (h) The really neat trick of PCR, however, is to do all of this within a single reaction vessel to which one has to add the various necessary ingredients only oncethereby repeated rounds of synthesis may be effected simply by heating and cooling the reaction vessel (i) The key step in the development of this technique, therefore, was the isolation and use of a heatresistant DNA polyermase (Tac DNA polymerase) (j) A key advantage that PCR has over the conventional cloning of DNA is that PCR may be initiated using only very small, impure samples of DNA (k) [PCR, polymerase chain reaction, thermocycler (Google)] [index] (139) Analysis of cloned DNA (a) The manipulation of DNA does not end with its cloning (b) A number of methods may be employed to analyze the cloned DNA as well as to compare that DNA with other samples of DNA (c) Among these many samples of DNA analysis include: (i) Southern blotting (ii) Restriction fragment length polymorphism (RFLP) (iii) In situ hybridization (iv) DNA sequencing (v) Etc. (d) Some of these methods will be considered below (e) [analysis of cloned DNA (Google Search)] [index] (140) Gel electrophoresis (a) One of the most useful techniques used for studying macromolecules (particularly proteins and nucleic acids) is gel electrophoresis (b) Gel electrophoresis is a method of molecule (or complex) separation in which molecules move within a gel-like medium (think jello or, even better, the agar found in a petri dish) (c) In gel electrophoresis charged molecules are pulled through the gel (typically consisting of the polymer polyacrylamide or of purified agar known as agarose) (i) Larger or more diffuse molecules move more slowly because they tend to get hung up in the gel matrix

(g)

Molecules with greater charges also move faster because it is an electric voltage that is employed to pull the molecules through the gel matrix (d) Often to negate charge differences between molecules as well as shape differences (e.g., as when comparing proteins) samples are first mixed with a charged detergent (SDS, a detergent you will typically find in shampoos) that serves to both denature and to even out and increase the amount of negative charge displayed by individual molecules (e) See Methods: Gel Electrophoresis of Macromolecules found on page 374 (f) [gel electrophoresis, SDS-PAGE (Google Search)] [index] (141) Southern blotting (a) Once you have DNA molecules separated on a gel it is possible to identify molecules that carry specific sequences (b) This identification is accomplished via the denaturation (separation of individual DNA strands) and then transfer of the DNA found in the gel to a kind of paper that is laid against the gel (c) The DNA now found on the paper is probed with tagged DNA fragments that, ideally, only bind to specific bands of DNA now found on the paper (d) The tagged fragments (e.g., radioactively tagged) are then visualized and the location or presence of the DNA on the original gel is inferred from its location or presence on the paper (e) See Methods: Restriction Fragment Analysis by Southern Blotting found on page 375 (f) [Southern blotting (Google Search)] [index] (142) Restriction fragment length polymorphism (RFLP) (a) RFLP analysis, along with PCR, was introduced to Americans particularly during the O. J. Simpson trial (b) The location of restriction sites in a genome occurs fairly randomly, and can differ from person to person essentially as allelic (mutational; nucleotide-sequence) difference between individuals (in general we call differences in nucleotide sequences at specific loci found within populations polymorphisms, in its originally meaning implying differences in the morphologies of individuals making up a population, implying genotypic differences) (c) As a consequence of these differences between individuals in the location of specific restriction sites, the distance between sites will vary, thus length of restriction fragments produced by digesting an individuals genome using specific restriction enzymes will also vary

(ii)

The variation between individuals is called restriction fragment length polymorphism (e) Since the nucleotide sequence of nearly every individual is unique, the RFLPs of each individual are also unique, and thus RFLP analysis may be employed to forensically distinguish individuals (hence the synonymous term, DNA fingerprinting) (f) RFLP analysis these days is employed for just about every population study you can imagine, used in everything from agriculture to forensics to wildlife biology (g) See Methods: Restriction Fragment Analysis by Southern Blotting found on page 375 (h) [RFLP, restriction fragment length polymorphism (Google Search)] [index] (143) In situ hybridization (a) Once you have a large amount of cloned (or PCRed) DNA, you can tag it either radioactively or fluorescently and then observe where on an intact chromosome that DNA binds (b) This is useful both for mapping cloned genes to regions on parent chromosome, or for identifying and locating similar genes found among different species (c) [in situ hybridization (Google Search)] [index] (144) DNA sequencing (a) Ultimately genotype consists of sequences of DNA nucleotides (or RNA nucleotides in the case of RNA viruses) (b) To determine genotype, one must determine the sequence of nucleotides and this is accomplished via a variety of techniques of DNA sequencing (c) These techniques involve DNA polymerization that is primed by specific, in vitro-synthesized oligonucleotides (short single-stranded DNA polymers) that serve to start the polymerization step at a certain point (essentially at the beginning of the to-be-determined nucleotide sequence) (d) Typically these oligonucleotides are tagged (e.g., radioactively or, more recently, fluorescently) such that the DNA strand that results from the DNA polymerization may be later visualized (e) Also required is some means of chain termination that is specific for individual bases plus the separation of DNAs to form single-stranded products (f) In this manner one ends up with a series of single-stranded DNAs that are tagged at one end for visualization and are of a specific length that is dependent upon where chain termination occurred (i.e., tag + primer + DNA chain/polymer + terminating nucleotide, found together in that order)

(d)

These DNAs are then separated via gel electrophoresis such that shorter DNAs (those that are cleaved earlier, i.e., have shorter DNA chains/polymers) migrate farther than longer DNAs (those that are cleaved later, i.e., have longer DNA chains/polymers) (h) By comparing the migration of DNAs in which chain termination occurred at A, T, G, or C nucleotides (typically one lane in a gel for each terminating nucleotide), one can infer the order that these nucleotides are found in the original DNA molecule (and for those of you who really get what I am saying but at this point are crying, Foul! please note that conditions are set up so that chain termination occurs with sufficiently low probability during the sequencing reaction that the polymerization of relatively long DNAs is possible, i.e., rather than each reaction terminating at the first A, T, G, or C found in a sequence) (i) See Methods: Sequencing of DNA by the Sanger Method found on page 378 (j) [DNA sequencing, Sanger method (Google Search)] [DNA sequencing links (MicroDude)] [index] (145) Vocabulary [index] (a) Analysis of cloned DNA (b) Biotechnology (c) cDNA (d) Cloning vector (e) Complementary DNA (f) DNA ligase (g) DNA sequencing (h) Expression vector (i) Gel electrophoresis (j) Gene cloning (k) Genetic engineering (l) Identification of the proper clones (m) In situ hybridization (n) Molecular biology (o) Molecular techniques (p) PCR (q) Polyermase chain reaction (r) Recombinant DNA (s) Restriction endonucleases (t) Restriction enzymes (u) Restriction fragment (v) Restriction fragment length polymorphism (w) Restriction site (x) RFLP (y) Southern blotting (z) Sticky ends (aa) Subcloning

(g)