Topic:Introduction to Genetics

Subject: Principles of Genetics Unit 1

The two kinds of nucleic acid, deoxyribonucleic acid(DNA) and ribonucleic acid (RNA), differ from oneanother in chemical composition and structure.

In procaryotic and eucaryotic cells, DNA serves as therepository for genetic information.

DNA is associated with basic proteins in the cell. Ineucaryotes these are special histone proteins, whereasin procaryotes nonhistone proteins are complexedwith DNA.

The flow of genetic information usually proceeds fromDNA through RNA to protein.

A protein’s amino acid sequence reflects thenucleotide sequence of its mRNA.

This messenger is a complementary copy of a portionof the DNA genome.

DNA replication is a very complex process involving avariety of proteins and a number of steps.

It is designed to operate rapidly while minimizingerrors and correcting those that arise when the DNAsequence is copied.

Genetic information is contained in the nucleotidesequence of DNA (and sometimes RNA).

When a structural gene directs the synthesis of apolypeptide, each amino acid is specified by a tripletcodon.

A gene is a nucleotide sequence that codes for apolypeptide, tRNA, or rRNA.

Most bacterial genes have at least four major parts,each with different functions: promoters, leaders,coding regions, and trailers.

Mutations are stable, heritable alterations in the genesequence and usually, but not always, producephenotypic changes. Nucleic acids are altered inseveral different ways, and these mutations may beeither spontaneous or induced by chemical mutagensor radiation.

It is extremely important to keep the nucleotidesequence constant, and microorganisms have severalrepair mechanisms designed to detect alterations inthe genetic material and restore it to its original state.

Often more than one repair system can correct aparticular type of mutation.

Despite these efforts some alterations remainuncorrected and provide material and opportunity forevolutionary change.

But the most important qualification of bacteriafor genetic studies is

their extremely rapid rate of growth. . . . a single E. colicell will grow overnight into a visible colony containingmillions of cells, even under relatively poor growthconditions.

Thus, genetic experiments on E. coli usually last oneday, whereas experiments on corn, for example, takemonths.

It is no wonder that we know so much more about thegenetics of E. coli than about the genetics of corn, eventhough we have been studying corn much longer.

—R.F.Weaver and P .W.Hedrick

DNA as Genetic Material

The early work of Fred Griffith in 1928 on the transferof virulence in the pathogen Streptococcuspneumoniae set the stage for the research that firstshowed that DNA was the genetic material.

Griffith found that if he boiled virulent bacteria andinjected them into mice, the mice were not affectedand no pneumococci could be recovered from theanimals. When he injected a combination of killedvirulent bacteria and a living nonvirulent strain, themice died; moreover, he could recover living virulentbacteria from the dead mice.

Griffith called this change of nonvirulent bacteria intovirulent pathogens transformation.

Oswald T. Avery and his colleagues then set out to discoverwhich constituent in the heat-killed virulent pneumococciwas responsible for Griffith’s transformation.

These investigators selectively destroyed constituents inpurified extracts of virulent pneumococci, using enzymesthat would hydrolyze DNA, RNA, or protein.

They then exposed nonvirulent pneumococcal strains tothe treated extracts. Transformation of the nonvirulentbacteria was blocked only if the DNA was destroyed,suggesting that DNA was carrying the informationrequired for transformation.

The publication of these studies by O. T. Avery, C. M.MacLeod, and M. J. McCarty in 1944 provided the firstevidence that Griffith’s transforming principle was DNAand therefore that DNA carried genetic information.

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Griffith’s Transformation Experiments. (a) Micedied of pneumonia when injected with pathogenicstrains of S pneumococci, which have a capsule andform smooth-looking colonies.

(b) Mice survived when injected with anonpathogenic strain of R pneumococci, whichlacks a capsule and forms rough colonies.

(c) Injection with heat-killed strains of Spneumococci had no effect.

(d) Injection with a live R strain and a heat-killedS strain gave the mice pneumonia, and live S strainpneumococci could be isolated from the dead mice.

Experiments on the Transforming Principle.Summary of the experiments of Avery, MacLeod, andMcCarty on the transforming principle. DNA alonechanged R to S cells, and this effect was lost when theextract was treated with deoxyribonuclease.

Thus DNA carried the genetic information requiredfor the R to S conversion or transformation.

Some years later (1952), Alfred D. Hershey and MarthaChase performed several experiments that indicatedthat DNA was the genetic material in the T2bacteriophage.

Some luck was involved in their discovery, for thegenetic material of many viruses is RNA and theresearchers happened to select a DNA virus for theirstudies.

Imagine the confusion if T2 had been an RNA virus!The controversy surrounding the nature of geneticinformation might have lasted considerably longerthan it did.

Hershey and Chase made the virus DNA radioactivewith 32P or labeled the viral protein coat with 35S

They mixed radioactive bacteriophage with E. coli andincubated the mixture for a few minutes.

The suspension was then agitated violently in aWaring blender to shear off any adsorbedbacteriophage particles. After centrifugation,radioactivity in the supernatant and the bacterialpellet was determined.

They found that most radioactive protein was releasedinto the supernatant, whereas 32P DNA remainedwithin the bacteria.

Since genetic material was injected and T2 progenywere produced, DNA must have been carrying thegenetic information for T2.

Subsequent studies on the genetics of viruses andbacteria were largely responsible for the rapiddevelopment of molecular genetics.

Furthermore, much of the new recombinant DNAtechnology has arisen from recent progress in bacterialand viral genetics.

Research in microbial genetics has had a profoundimpact on biology as a science and on the technologythat affects everyday life.

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The Hershey-Chase Experiment.

(a) When E. coli was infected with a T2 phagecontaining 35S protein, most of the radioactivityremained outside the host cell.

(b) When a T2 phage containing 32P DNA was mixedwith the host bacterium, the radioactive DNA wasinjected into the cell and phages were produced. ThusDNA was carrying the virus’s genetic information.

Biologists have long recognized a relationship betweenDNA, RNA, and protein, and this recognition hasguided a vast amount of research over the past decades.DNA is precisely copied during its synthesis orreplication.

The expression of the information encoded in the basesequence of DNA begins with the synthesis of an RNAcopy of the DNA sequence making up a gene.

A gene is a DNA segment or sequence that codes for apolypeptide, an rRNA, or a tRNA.

Although DNA has two complementary strands, only thetemplate strand is copied at any particular point on DNA.

If both strands of DNA were transcribed, two differentmRNAs would result and cause genetic confusion.

Thus the sequence corresponding to a gene is located onlyon one of the two complementary DNA strands.

Different genes may be encoded on opposite strands.This process of DNA-directed RNA synthesis is calledtranscription because the DNA base sequence isbeing written into an RNA base sequence.

The RNA that carries information from DNA anddirects protein synthesis is messenger RNA (mRNA).

The last phase of gene expression is translation orprotein synthesis.

The genetic information in the form of an mRNAnucleotide sequence is translated and governs thesynthesis of protein.

Thus the amino acid sequence of a protein is a directreflection of the base sequence in mRNA.

In turn the mRNA nucleotide sequence is acomplementary copy of a portion of the DNA genome.

Relationships between DNA, RNA, and ProteinSynthesis. This conceptual framework is sometimes called the CentralDogma.

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Nucleic Acid Structure

The structure and synthesis of purine and pyrimidinenucleotides are introduced in chapter. These nucleotidescan be combined to form nucleic acids of two kinds.

Deoxyribonucleic acid (DNA) contains the 2′-deoxyribonucleosides of adenine, guanine, cytosine, andthymine.

Ribonucleic acid (RNA) is composed of theribonucleosides of adenine, guanine, cytosine, anduracil (instead of thymine). In both DNA and RNA,nucleosides are joined by phosphate groups to form longpolynucleotide chains.

The differences in chemical composition between thechains reside in their sugar and pyrimi-dine bases: DNAhas deoxyribose and thymine; RNA has ribose and uracil inplace of thymine.

(a) A diagram showing the relationships of various nucleic acid components. Combination of a purine or pyrimidine base with ribose or deoxyribose gives a nucleoside (a ribonucleoside or deoxyribonucleoside). A nucleotide contains a nucleoside and one or more phosphoric acid molecules. Nucleic acids result when nucleotides are connected together in polynucleotide chains.

(b) Examplesc of nucleosides—the purine nucleoside adenosine and the pyrimidine deoxynucleoside 2′-deoxycytidine. The carbons of nucleoside sugars are indicated by numbers with primes.

(c) A segment of a polynucleotide chain showing two nucleosides, deoxyguanosine and thymidine, connected by a phosphodiester linkage between the 3′ and 5′-carbons of adjacent deoxyribose sugars.

DNA Structure

Deoxyribonucleic acids are very large molecules,usually composed of two polynucleotide chains coiledtogether to form a double helix 2.0 nm in diameter.

Each chain contains purine and pyrimidinedeoxyribonucleosides joined by phosphodiesterbridges.

That is, two adjacent deoxyribose sugars areconnected by a phosphoric acid molecule esterified toa 3′- hydroxyl of one sugar and a 5′-hydroxyl of theother.

Purine and pyrimidine bases are attached to the 1′-carbon of the deoxyribose sugars and extend towardthe middle of the cylinder formed by the two chains.

They are stacked on top of each other in the center,one base pair every 0.34 nm. The purine adenine (A) isalways paired with the pyrimidine thymine (T) by twohydrogen bonds. The purine guanine (G) pairs withcytosine (C) by three hydrogen bonds.

This AT and GC base pairing means that the twostrands in a DNA double helix are complementary.That is, the bases in one strand match up with thoseof the other according to the base pairing rules.

Because the sequences of bases in thesestrandsencode genetic information, considerable effort hasbeen devoted to determining the base sequences ofDNA and RNA from many microorganisms

The two polynucleotide strands fit together much like thepieces in a jigsaw puzzle because of complementary basepairing. Inspection of figure depicting the B form ofDNA (probably the most common form in cells), showsthat the two strands are not positioned directly oppositeone another in the helical cylinder.

Therefore, when the strands twist about one another, awide major groove and narrower minor groove areformed by the backbone.

Each base pair rotates 36° around the cylinder with respectto adjacent pairs so that there are 10 base pairs per turn ofthe helical spiral.

Each turn of the helix has a vertical length of 3.4 nm. Thehelix is right-handed—that is, the chains turncounterclockwise as they approach a viewer looking downthe longitudinal axis.

The two backbones are antiparallel or run in oppositedirections with respect to the orientation of theirsugars.

One end of each strand has an exposed 5′-hydroxylgroup, often with phosphates attached, whereas theother end has a free 3′-hydroxyl group.

If the end of a double helix is examined, the5′ end ofone strand and the 3′ end of the other are visible.

In a given direction one strand is oriented 5′ to 3′ andthe other, 3′ to 5′

(a) A space-filling model of the B form of DNA with the base pairs, major groove, and minor groove shown. The backbone phosphate groups, shown in color, spiral around the outside of the helix.

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(b) A diagrammatic representation of the double helix. The backboneconsists of deoxyribose sugars (S) joined by phosphates (P) in phosphodiesterbridges. The arrows at the top and bottom of the chains point in the 5′ to 3′direction. The ribbons represent the sugar phosphate backbones.

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DNA Base Pairs. DNA complementary base pairing showing the hydrogen bonds

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RNA Structure

Besides differing chemically from DNA, ribonucleicacid is usually single stranded rather than doublestranded like most DNA.

An RNA strand can coil back on itself to form ahairpin-shaped structure with complementary basepairing and helical organization.

Cells contain three different types of RNA—messengerRNA, ribosomal RNA, and transfer RNA—that differfrom one another in function, site of synthesis ineucaryotic cells, andstructure.

The Organization of DNA in Cells

Although DNA exists as a double helix in bothprocaryotic and eucaryotic cells, its organizationdiffers in the two cell types.

DNA is organized in the form of a closed circle inalmost all procaryotes (the chromosome of Borrelia isa linear DNA molecule).

This circular double helix is further twistedintosupercoiled DNA and is associated with basicproteins but not with the histones found complexedwith almost all eucaryotic DNA.

These histone like proteins do appear to help organizebacterial DNA into a coiled chromatinlike structure.

DNA is much more highly organized in eucaryoticchromatin and is associated with a variety of proteins,the most prominent of which are histones.

These are small, basic proteins rich in the aminoacids lysine and/or arginine. There are five types ofhistones in almost all eucaryotic cells studied: H1,H2A, H2B, H3, and H4.

Eight histone molecules (two each of H2A, H2B, H3,and H4) form an ellipsoid about 11 nm long and 6.5 to7 nm in diameter.

DNA coils around the surface of the ellipsoidapproximately 13 4 turns or 166 base pairs beforeproceeding on to the next. This complex of histonesplus DNA is called a nucleosome.

Thus DNA gently isolated from chromatin lookslike a string of beads. The stretch of DNA between thebeads or nucleosomes, the linker region, varies inlength from 14 to over 100 base pairs.

Histone H1 appears to associate with the linker regionsto aid the folding of DNA into more complexchromatin structures.

When folding reaches a maximum, the chromatin takesthe shape of the visible chromosomes seen ineucaryotic cells during mitosis and meiosis

Nucleosome Internal Organization and Function.(a) The nucleosome core particle is a histone octamer surrounded by the 146 base pair DNA helix (brown and turquoise). The octamer is a disk-shaped structure composed of two H2A-H2B dimers and two H3-H4 dimers. The eight histone proteins are coloreddifferently: blue, H3; green, H4; yellow, H2A; and red, H2B. Histone proteins interact with the backbone of the DNA minor groove. The DNA double helix circles the histoneoctamer in a lefthanded helical path.

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Medicine

In medicine, genetic engineering has been used tomass-produce insulin, human growth hormones,follistim (for treating infertility), human albumin,monoclonal antibodies, antihemophilic factors,vaccines and many other drugs. Vaccination generallyinvolves injecting weak, live, killed or inactivatedforms of viruses or their toxins into the person beingimmunized. Genetically engineered viruses are beingdeveloped that can still confer immunity, but lack theinfectious sequences. Mouse hybridomas, cells fusedtogether to create monoclonal antibodies, have beenhumanised through genetic engineering to createhuman monoclonal antibodies. Genetic engineeringhas shown promise for treating certain forms of cancer.

Genetic engineering is used to create animal models ofhuman diseases. Genetically modified mice are themost common genetically engineered animal model.They have been used to study and model cancer (theoncomouse), obesity, heart disease, diabetes, arthritis,substance abuse, anxiety, aging and Parkinson disease.Potential cures can be tested against these mousemodels. Also genetically modified pigs have been bredwith the aim of increasing the success of pig to humanorgan transplantation.

Gene therapy is the genetic engineering of humans byreplacing defective human genes with functional copies.This can occur in somatic tissue or germline tissue. If thegene is inserted into the germline tissue it can be passeddown to that person's descendants. Gene therapy has beensuccessfully used to treat multiple diseases, including X-linked SCID, chronic lymphocytic leukemia (CLL), andParkinson's disease. In 2012, Glybera became the first genetherapy treatment to be approved for clinical use in eitherEurope or the United States after its endorsement by theEuropean Commission. There are also ethical concernsshould the technology be used not just for treatment, butfor enhancement, modification or alteration of a humanbeings' appearance, adaptability, intelligence, character orbehavior. The distinction between cure and enhancementcan also be difficult to establish. Transhumanists considerthe enhancement of humans desirable.

Research Knockout mice Human cells in which some proteins are fused with green

fluorescent protein to allow them to be visualised Genetic engineering is an important tool for natural

scientists. Genes and other genetic information from awide range of organisms are transformed into bacteria forstorage and modification, creating genetically modifiedbacteria in the process. Bacteria are cheap, easy to grow,clonal, multiply quickly, relatively easy to transform andcan be stored at -80 °C almost indefinitely. Once a gene isisolated it can be stored inside the bacteria providing anunlimited supply for research.

Organisms are genetically engineered to discover thefunctions of certain genes. This could be the effect on thephenotype of the organism, where the gene is expressed orwhat other genes it interacts with. These experimentsgenerally involve loss of function, gain of function, trackingand expression.

Loss of function experiments, such as in a geneknockout experiment, in which an organism is engineeredto lack the activity of one or more genes. A knockoutexperiment involves the creation and manipulation of aDNA construct in vitro, which, in a simple knockout,consists of a copy of the desired gene, which has beenaltered such that it is non-functional. Embryonic stem cellsincorporate the altered gene, which replaces the alreadypresent functional copy. These stem cells are injected intoblastocysts, which are implanted into surrogate mothers.This allows the experimenter to analyze the defects causedby this mutation and thereby determine the role ofparticular genes. It is used especially frequently indevelopmental biology. Another method, useful inorganisms such as Drosophila (fruit fly), is to inducemutations in a large population and then screen theprogeny for the desired mutation. A similar process can beused in both plants and prokaryotes.

Gain of function experiments, the logicalcounterpart of knockouts. These are sometimesperformed in conjunction with knockout experimentsto more finely establish the function of the desiredgene. The process is much the same as that inknockout engineering, except that the construct isdesigned to increase the function of the gene, usuallyby providing extra copies of the gene or inducingsynthesis of the protein more frequently.

Tracking experiments, which seek to gaininformation about the localization and interaction ofthe desired protein. One way to do this is to replacethe wild-type gene with a 'fusion' gene, which is ajuxtaposition of the wild-type gene with a reportingelement such as green fluorescent protein (GFP) thatwill allow easy visualization of the products of thegenetic modification. While this is a useful technique,the manipulation can destroy the function of the gene,creating secondary effects and possibly calling intoquestion the results of the experiment. Moresophisticated techniques are now in development thatcan track protein products without mitigating theirfunction, such as the addition of small sequences thatwill serve as binding motifs to monoclonal antibodies.

Expression studies aim to discover where and whenspecific proteins are produced. In these experiments,the DNA sequence before the DNA that codes for aprotein, known as a gene's promoter, is reintroducedinto an organism with the protein coding regionreplaced by a reporter gene such as GFP or an enzymethat catalyzes the production of a dye. Thus the timeand place where a particular protein is produced canbe observed. Expression studies can be taken a stepfurther by altering the promoter to find which piecesare crucial for the proper expression of the gene andare actually bound by transcription factor proteins;this process is known as promoter bashing.

One of the best-known and controversial applicationsof genetic engineering is the creation and use ofgenetically modified crops or genetically modifiedorganisms, such as genetically modified fish, which areused to produce genetically modified food andmaterials with diverse uses. There are four main goalsin generating genetically modified crops.

One goal, and the first to be realized commercially, isto provide protection from environmental threats,such as cold (in the case of Ice-minus bacteria), orpathogens, such as insects or viruses, and/or resistanceto herbicides. There are also fungal and virus resistantcrops developed or in development. They have beendeveloped to make the insect and weed managementof crops easier and can indirectly increase crop yield.

Another goal in generating GMOs is to modify the qualityof produce by, for instance, increasing the nutritional valueor providing more industrially useful qualities orquantities. The Amflora potato, for example, produces amore industrially useful blend of starches. Cows have beenengineered to produce more protein in their milk tofacilitate cheese production. Soybeans and canola havebeen genetically modified to produce more healthy oils.

Another goal consists of driving the GMO to producematerials that it does not normally make. One example is"pharming", which uses crops as bioreactors to producevaccines, drug intermediates, or drug themselves; theuseful product is purified from the harvest and then usedin the standard pharmaceutical production process. Cowsand goats have been engineered to express drugs and otherproteins in their milk, and in 2009 the FDA approved adrug produced in goat milk.

Another goal in generating GMOs, is to directly improveyield by accelerating growth, or making the organism morehardy (for plants, by improving salt, cold or droughttolerance). Some agriculturally important animals havebeen genetically modified with growth hormones toincrease their size.

The genetic engineering of agricultural crops can increasethe growth rates and resistance to different diseases causedby pathogens and parasites. This is beneficial as it cangreatly increase the production of food sources with theusage of fewer resources that would be required to host theworld's growing populations. These modified crops wouldalso reduce the usage of chemicals, such as fertilizers andpesticides, and therefore decrease the severity andfrequency of the damages produced by these chemicalpollution.

Ethical and safety concerns have been raised around theuse of genetically modified food. A major safety concernrelates to the human health implications of eatinggenetically modified food, in particular whether toxic orallergic reactions could occur. Gene flow into related non-transgenic crops, off target effects on beneficial organismsand the impact on biodiversity are importantenvironmental issues. Ethical concerns involve religiousissues, corporate control of the food supply, intellectualproperty rights and the level of labeling needed ongenetically modified products. BioArt andentertainment

Genetic engineering is also being used to create BioArt.Some bacteria have been genetically engineered to createblack and white photographs.

Genetic engineering has also been used to create noveltyitems such as lavender-colored carnations, blue roses, andglowing fish.

References

Fig- 1,3,4 http://classroom.sdmesa.edu/eschmid/Lecture6-Microbio.htm

Fig 2

http://www.accessexcellence.org/RC/VL/GG/hershey.php

Fig 5

http://geneed.nlm.nih.gov/topic_subtopic.php?tid=15&sid=16

Fig 6

Principles of Biochemistry by Lehninger

Fig 7 http://www.nature.com/nature/journal/v389/n6648/full/389251a0.html

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