Heredity, Genes, and DNA *All living things are able to reproduce (inherit the genetic information from their parents) *All cells arise from pre-existing cells (genetic material must be replicated & passed from parent to progeny cell at each cell division)Genes – a pair of inherited factors which determines a trait Allele- one gene copy specifying each trait is inherited from each parent Genotype- genetic composition (eg. Bb) Phenotype- physical appearance (eg. straight hair) Chromosomes- carrier of genes * cells of higher plants and animals are DIPLOID - containing 2 copies of each chromosome * sperm and egg cell containing only 1 copy of each chromosome- HAPLOID
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Figure 3.3. Gene segregationand linkage (A) Segregation of
two hypothetical genes forshape ( A/a = square/round)and color (B/b = red/blue)located on differentchromosomes. (B) Linkage oftwo genes located on the same
Figure 3.5. A genetic map Three genes arelocalized on a hypothetical chromosome
based on frequencies of recombinationbetween them (1% recombination betweena and b; 3% between b and c; 4% between a and c). The frequencies of recombinationare approximately proportional to thedistances between genes on the
In molecular terms, a gene commonly is defined as the entire nucleicacid sequence that is necessary for the synthesis of a functional polypeptide.
Identification of DNA as the Genetic Material:
Bacterial genes are made of DNA
Figure 4-2. Experimental demonstration thatDNA is the genetic material. Theseexperiments, carried out in the 1940s, showedthat adding purified DNA to a bacteriumchanged its properties and that this change was
faithfully passed on to subsequent generations.Two closely related strains of the bacteriumStreptococcus pneumoniae differ from each otherin both their appearance under the microscopeand their pathogenicity. One strain appearssmooth (S) and causes death when injected intomice, and the other appears rough (R) and isnonlethal. (A) This experiment shows that a
substance present in the S strain can change (ortransform) the R strain into the S strain and thatthis change is inherited by subsequentgenerations of bacteria. (B) This experiment, inwhich the R strain has been incubated withvarious classes of biological molecules obtainedfrom the S strain, identifies the substance as
In 1944 Oswald Avery, Colin MacLeod, and Maclyn McCarty establishedthat the transforming principle was DNA
Figure 1.3. The transformingprinciple is DNA. Avery and
his colleagues showed that thetransforming principle isunaffected by treatment with aprotease or a ribonuclease, butis inactivated by treatment witha deoxyribonuclease
Virus genes are made of DNA Figure 1.4. Bacteriophages areviruses that infect bacteria. (A) Thestructure of a head-and-tailbacteriophage such as T2. The
DNA genome of the phage iscontained in the head part of theprotein capsid. (B) The infectioncycle. After injection into anEscherichia coli bacterium, the T2phage genome directs synthesis ofnew phages. For T2, the infectioncycle takes about 20 minutes at 37°C and ends with lysis of the celland release of 250–300 new phages.This is the lytic infection cycle.
Some phages, such as λ, can alsofollow a lysogenic infection cycle,in which the phage genomebecomes inserted into the bacterialchromosome and remains there, inquiescent form, for several
generations of the bacterium(Section 4.2.1).(Hershey and Chase, 1952)
DNA was discovered in 1869 by Johann Friedrich Miescher, a Swiss biochemistworking in Tubingen, Germany (1st using crude extract from human WBC then apurified one from salmon sperm)
Deoxyribonucleic acid (DNA) is the storehouse, or cellular library, that contains allthe information required to build the cells and tissues of an organism.
The full chemical names of the four nucleotides that polymerize to make DNAare:2′-deoxyadenosine 5′-triphosphate2′-deoxycytidine 5′-triphosphate2′-deoxyguanosine 5′-triphosphate2′-deoxythymidine 5′-triphosphate
DNA is a linear, unbranched polymer in which the monomeric subunits are fourchemically distinct nucleotides that can be linked together in any order in chainshundreds, thousands or even millions of units in length.
Each nucleotide in a DNA polymer is made up of three components:
The double helix- according to Watson, their work was a desperate race againstAmerican biochemist, Linus Pauling , who initially proposed
an incorrect triple helix model-Rosalind Franklin, whose X-ray diffraction studies provided the bulk ofthe experimental data in support of the double helix and who was herselfvery close to solving the structure.
-double helix, discovered by Watson and Crick on Saturday 7 March 1953,
was the single most important breakthrough in biology during the 20thcentury.
The evidence that led to the double helix
Watson and Crick used four types of information to deduce the double helixstructure:
1. Biophysical data of various kinds. The water content of DNA fibers wasparticularly important because it enabled the density of the DNA in a fiber tobe estimated. The number of strands in the helix and the spacing between thenucleotides had to be compatible with the fiber density. Pauling's triple helixmodel was based on an incorrect density measurement which suggested thatthe DNA molecule was more closely packed than it actually is.
2. X-ray diffraction patterns (Section 9.1.3), most of which were produced byRosalind Franklin of Kings College, London, and which revealed the helical nature
of the structure and indicated some of the key dimensions within the helix.
3. The base ratios, which had been discovered by Erwin Chargaff of ColumbiaUniversity, New York. Chargaff carried out a lengthy series of chromatographicstudies of DNA samples from various sources and showed that, although thevalues are different in different organisms, the amount of adenine is always the
same as the amount of thymine, and the amount of guanine equals the amount ofcytosine ( Figure 1.10 ). These base ratios led to the base-pairing rules, which werethe key to the discovery of the double helix structure.
4. Model building , which was the only major technique that Watson and Crickmade use of themselves. Scale models of possible DNA structures enabled therelative positioning of the various atoms to be checked, to ensure that pairs ofgroups that formed bonds were not too far apart, and that other groups were not soclose together as to interfere with one another.
Figure 1.11. The double helix structure ofDNA. (A) Two representations of thedouble helix. On the left the structure isshown with the sugar-phosphate‘backbones' of each polynucleotidedrawn as a red ribbon with the base
pairs in black. On the right the chemicalstructure for three base pairs is given. (B)A base-pairs with T, and G base-pairswith C. The bases are drawn in outline,with the hydrogen bonding indicated bydotted lines. Note that a G-C base pair
has three hydrogen bonds whereas an A-T base pair has just two. The structuresin part (A) are redrawn from Turner etal. (1997) (left) and Strachan and Read(1999) (right).
The key features of the double helix- double helix is right-handed- two strands run in opposite directions- helix is stabilized by two types of chemical interaction:
1. Base-pairing between the two strands involves the formation ofhydrogen bonds between an adenine on one strand and athymine on the other strand, or between a cytosine and aguanine
2. Base-stacking, sometimes called - interactions, involveshydrophobic interactions between adjacent base pairs and adds
stability to the double helix once the strands have been broughttogether by base-pairing. These hydrophobic interactions arisebecause the hydrogen-bonded structure of water forceshydrophobic groups into the internal parts of a molecule.
The double helix has structural flexibility
-double helix described by Watson and Crick, is called the B-form of DNA-that genomic DNA molecules are not entirely uniform in structure. This ismainly because each nucleotide in the helix has the flexibility to take upslightly different molecular shapes
-Rotations within individual nucleotides therefore lead to major changes in theoverall structure of the helix.
Figure 3.10. Colinearity of genesand proteins A series of mutations(arrowheads) were mapped in theE. coli gene encoding tryptophansynthetase (top line). The amino
acid substitutions resulting fromeach of the mutations was thendetermined by sequence analysisof the proteins of mutant bacteria(bottom line). These studiesrevealed that the order ofmutations in DNA was the sameas the order of amino acidsubstitutions in the encodedprotein.
-the correspondence between nucleotide triplets and amino acids in proteins.-it is a triplet code (43) or 64 codons-of the 64 codons, 61 specify an amino acid; the remaining three (UAA, UAG,and UGA) are stop codons that signal the termination of protein synthesis.-the code is degenerate(meaning, many amino acids are specified by more than
Figure 3.14. The triplet UUU encodes phenylalanine In vitro translation ofa synthetic RNA consisting of repeated uracils (a poly-U template) results inthe synthesis of a polypeptide containing only phenylalanine.
Molecular cloning is to insert a DNA fragment of interest (e.g., a segment of humanDNA) into a DNA molecule (called a vector) that is capable of independent
Figure 3.25. Automated DNAsequencing Four separatesequencing reactions areperformed, each containing onechain-terminatingdideoxynucleotide and a primerlabeled with a distinct fluorescenttag. The products are then pooledand subjected to gelelectrophoresis. As the DNAstrands migrate through the gel,they pass through a laser beam thatexcites the fluorescent label. Theemitted light is detected by aphotomultiplier, which is
connected to a computer thatcollects and analyzes the data.
Use to determine completegenome sequences of bacteria,yeast, C. elegans, and Drosophila,
Figure 3.26. Expression ofcloned genes in bacteria Expression vectors containpromoter sequences (pro) thatdirect transcription of inserted
DNA in bacteria and sequencesrequired for binding of mRNAto bacterial ribosomes (Shine-Delgarno [SD] sequences). Aeukaryotic cDNA insertedadjacent to these sequences can
be efficiently expressed in E.coli, resulting in production ofeukaryotic proteins intransformed bacteria
Figure 3.27. Amplification ofDNA by PCR The region ofDNA to be amplified is flankedby two sequences used to primeDNA synthesis. The startingdouble-stranded DNA is heatedto separate the strands and thencooled to allow primers (usually
oligonucleotides of 15 to 20bases) to bind to each strand ofDNA. DNA polymerase fromThermus aquaticus (Taq polymerase) is used tosynthesize new DNA strands
starting from the primers,resulting in the formation of twonew DNA molecules. Theprocess can be repeated formultiple cycles, each resulting ina twofold amplification of DNA.
Recombinant DNA libraries—collections of clones that contain all the genomicor mRNA sequences of a particular cell type
Genomic library -A collection of recombinant DNA clones that collectivelycontain the genome of an organismcDNA library -A collection of recombinant cDNA clones.
Variety of probes can be used for screening recombinant libraries:
1. a cDNA clone can be used as a probe to isolate thecorresponding genomic clone2. a gene cloned from one species (e.g., mouse) can be used to isolate a
related gene from a different species (e.g., human).3. aside from isolated DNA fragments, synthetic oligonucleotides can
be used as probes, enabling the isolation of genes on the basisof partial amino acid sequences of their encoded proteins.
4. use of antibodies as probes to screen expression libraries
-understanding the function of a gene, however, requires analysis of thegene within cells or intact organisms—not simply as a molecular clone inbacteria.
Genetic Analysis in Yeasts Figure 3.34. Cloning of yeast genes (A) A yeast vector.The vector contains a bacterial origin of replication (ori)and an ampicillin resistance gene ( Ampr), allowing it tobe propagated as a plasmid in E. coli. In addition, thevector contains a yeast origin of replication and amarker gene (LEU2), allowing the selection of
transformed yeast. The LEU2 gene encodes an enzymerequired for synthesis of the amino acid leucine, sotransformation of yeast strains lacking this enzyme canbe selected for by growth on medium lacking leucine.(B) Isolation of a yeast gene. A gene of interest isidentified by a temperature-sensitive mutation, whichallows yeast to grow at 25°C but not at 37°C. To isolatea clone of the gene, the temperature-sensitive yeasts aretransformed with a plasmid library containing acollection of genes encompassing the entire yeastgenome. All yeasts transformed by plasmid DNAs areable to grow on media lacking leucine at 25°C, but onlythose yeasts transformed by a plasmid carrying anormal copy of the gene of interest are able to grow at37°C. The desired plasmid can be isolated from
transformed yeasts that form colonies at thenonpermissive temperature.
Mutagenesis of Cloned DNAs-In classical genetic studies (e.g., in bacteria or yeasts), mutants are the key toidentifying genes and understanding their function by observing the alteredphenotype of mutant organisms.
Classical genetics (forward genetics)
Random mutagenesis Natural mutants
An altered phenotype is screened (genetic screening).