LEARNING UNIT

LEARNING UNIT 22
PROKARYOTIC GENOME
PROKARYOTIC GENOME SYSTEM
SYSTEM
• IN THIS LEARNING UNIT, WE WILL COVER THE FOLLOWING AREAS:
1. STRUCTURE & FUNCTIONS OF PROKARYOTIC CHROMOSOMES.
2. STRUCTURE & FUNCTIONS OF EXTRACHROMOSOMAL VECTOR
ELEMENTS (PLASMIDS)
3. STRUCTURE & FUNCTIONS OF TRANSPOSITIONAL ELEMENTS
(TRANSPOSONS).
4. PROKARYOTIC RECOMBINANT DNA MECHANISMS
• IN THIS LEARNING UNIT, NEW TERMINOLOGIES TO UNDERSTAND:
1. PROKARYOTIC GENOME SYSTEM - BACTERIAL CHROMOSOME,
PLASMIDS & TRANSPOSON.
2. PROKARYOTIC RECOMBINANT DNA – TRANSFORMATION,
TRANSDUCTION & CONJUGATION.
LEARNING UNIT
LEARNING UNIT 22
PROKARYOTIC GENOME
PROKARYOTIC GENOME SYSTEM
SYSTEM
• BY THE END OF TODAY’S LECTURE, STUDENTS SHOULD BE ABLE TO:
1. DEFINE THE CONCEPTS AND THE UNIQUENESS OF THE
PROKARYOTIC GENOME SYSTEM.
2. DESCRIBE THE DIFFERENCES BETWEEN THE EUKARYOTIC AND
PROKARYOTIC GENOME SYSTEM.
3. DIFFERENTIATE THE VARIOUS TYPES OF BACTERIAL
RECOMBINATION MECHANISMS.
4. EXPLAIN THE THEORIES AND STEPS IN BACTERIAL
RECOMBINATION MECHANISMS.
5. EVALUATE THE RELEVANCY OF BACTERIAL RECOMBINATION
MECHANISMS TO THE FIELD OF BIOTECHNOLGY.

INTRO
PROKARYOTIC GENOME
PROKARYOTIC GENOME SYSTEM
SYSTEM - INTRODUCTION
INTRODUCTION
• IN THE STUDY OF PROKARYOTIC GENOME SYSTEM, E. COLI IS THE MODEL
ORGANISM.
• IT MAKES AN EXCELLENT MODEL ORGANISM BECAUSE IT IS:
• INEXPENSIVE & SMALL - CAN HAVE A VERY LARGE POPULATION (EASILY 10 99
CELLS) IN ONE TUBE –GREAT STATISTICS
• READILY CULTURED (FAST-GROWING) – 20 MIN UNDER OPTIMAL CONDITIONS.
• SMALL GENOME (4300 GENES)
• HAPLOID AND REPRODUCES ASEXUALLY
• MOBILE GENETIC ELEMENTS (PLASMIDS AND PHAGES)
• RAPID EVOLUTION (10-7 MUTATIONS PER GENE PER REPLICATION.) - CAN BE
MUTAGENIZED TO INCREASE THE FREQUENCY OF MUTATIONS: DO NOT HAVE TO
RELY ON VERY RARE EVENTS
• BECAUSE GENOME IS TIGHTLY PACKED, YOU HAVE MORE (PROPORTIONALLY) OF
THE IMPORTANT STUFF TO STUDY
• PROKARYOTIC GENOME SYSTEM CONSIST OF THE FOLLOWING :
• BACTERIAL CHROMOSOME
• PLASMIDS
• TRANSPOSONS
BACTERIAL CHROMOSOME
BACTERIAL CHROMOSOMES
BACTERIAL
• WE NOW KNOW THE COMPLETE DNA SEQUENCES FOR OVER THIRTY
BACTERIAL SPECIES AND STRAINS.
• I.E. VIBRIO CHOLERAE, E. COLI, SACCHAROMYCIES CEREVISAE
• A BACTERIAL CELL, IS GENERALLY BETWEEN 1-3
µm LONG, MAY CONTAIN A DNA MOLECULE THAT
IS 1000 TIMES LONGER THAN ITS LENGTH.
• (MOST) PROKARYOTIC CHROMOSOMES CONSIST
OF ONE LONG, SINGLE MOLECULE OF DOUBLE
STRANDED, HELICAL, CIRCULAR STRUCTURE.
• DNA IS ARRANGED IN A DENSE CLUMP CALLED
NUCLEUS.
• IT IS MUCH SMALLER THAN EUKARYOTIC
CHROMOSOMES, AND ENCODES ABOUT 3,000
GENES (UP TO ABOUT 4,000).
• THE SIZE OF BACTERIA CHROMOSOMES RANGES
FROM 580 Kbp TO 10,000 Kbp.
• (ARCHAE CHROMOSOMES RANGE FROM 500 Kbp TO
5,800 Kbp).
BACTERIAL CHROMOSOMES
BACTERIAL
• BACTERIA HAVE SMALL, COMPACT GENOMES, AND NEED ABOUT 500 TO
5000 GENES TO CONDUCT THEIR LIVES
• THE SMALLEST GENOMES ARE IN THE MYCOPLASMA. FOR EXAMPLE, THE
COMPLETE M. GENITALIUM GENOME IS ONLY 580 Kbp AND IS PREDICTED TO
ENCODE 480 PROTEINS.

BASE PAIRS GENES

HUMAN MITOCHONDRION 16,569 37
MYCOPLASMA PNEUMONIAE 816,394 680
TREPONEMA PALLIDUM 1,138,011 1,039
METHANOCOCCUS JANNASCHII 1,664,970 1,783
STREPTOCOCCUS PNEUMONIAE 2,160,837 2,236
VIBRIO CHOLERAE 4,033,460 3,890
MYCOBACTERIUM TUBERCULOSIS 4,411,532 3,959
MYCOBACTERIUM LEPRAE 3,268,203 1,604
E. COLI 4,639,221 4,377
E. COLI O157:H7 5.44 X 106 5,416
SACCHAROMYCES CEREVISIAE 12,495,682 5,770
HUMANS 3.3 X 109 20,000–25,000
BACTERIAL CHROMOSOMES
BACTERIAL
• THE CHROMOSOME FORM GENETIC CIRCLE IN A SUPERCOILED DNA.
• BACTERIAL ENZYMES CALLED DNA GYRASE AND DNA TOPOISOMERASES ARE
ESSENTIAL IN THE UNWINDING, REPLICATION, AND REWINDING OF THE
CIRCULAR, SUPERCOILED PROKARYOTIC DNA.
• BECAUSE THEY ARE SMALL, BACTERIAL GENOMES ARE HIGHLY
ORGANIZED, FEW INTRONS ARE FOUND IN BACTERIA
• PROKARYOTES HAVE VERY LITTLE JUNK DNA (TYPICALLY LESS THAN 15% OF
THE GENOME) AND EUKARYOTES HAVE SUBSTANTIAL AMOUNTS OF JUNK DNA.
• TRANSCRIPTION OCCURS IN CYTOPLASM ALONG WITH TRANSLATION.
• SINCE PROKARYOTES ARE HAPLOID (HAVE ONLY ONE CHROMOSOME),
THERE IS NO MEIOSIS.
• IN ADDITION, BACTERIA MAY CONTAIN ACCESSORY "CHROMOSOMES"
CALLED PLASMIDS.

PLASMIDS
PLASMIDS
PLASMIDS
• PLASMIDS ARE INDEPENDENT EXTRA
GENETIC ELEMENTS FOUND IN BACTERIA AS
WELL AS IN SOME YEAST AND FUNGI.
• PLASMIDS ARE ALSO CALLED VECTORS.
• PLASMIDS ARE LIKE VIRUSES, BUT HAVE NO
EXTRACELLULAR PHASE.
• A TYPICAL PLASMID TYPICALLY HAVE
THREE IMPORTANT ELEMENTS:
• AN ORIGIN OF REPLICATION.
• A SELECTABLE MARKER GENE
• A CLONING SITE
• MOST PLASMIDS ARE CIRCULLAR, WITH A
COVALENTLY CLOSED CIRCULAR DOUBLE-
STRANDED DNA MOLECULES.
• USUALLY CARRY ONLY ONE OR A FEW GENES, CODING FOR SPECIAL
FUNCTION (E.G. ANTIBIOTIC RESISTANCE, VIRULENCE, CATABOLISM OF PHENOLIC
SUBSTANCES AND NITROGEN FIXATION.)
PLASMIDS
PLASMIDS
• PLASMIDS ARE REPLICATED BY THE SAME MACHINERY THAT REPLICATES
THE BACTERIAL CHROMOSOME.
• SOME PLASMIDS ARE COPIED AT ABOUT THE SAME
RATE AS THE CHROMOSOME, SO A SINGLE CELL HAVE
ONLY A SINGLE COPY OF THE PLASMID.
• OTHER PLASMIDS ARE COPIED AT A HIGH RATE AND A
SINGLE CELL MAY HAVE 50 OR MORE OF THEM.
• PLASMIDS ARE INHERITED WITH A HIGH DEGREE OF STABILITY.
• PLASMIDS ALSO ALLOW BACTERIA TO SHARE GENES RAPIDLY.
• PLASMIDS ARE NOT ESSENTIAL FOR THE BASIC OPERATION OF
BACTERIA, BUT THEY ENCODE A WIDE VARIETY OF GENETIC
DETERMINANTS, THAT PROVIDE SOME BENEFIT TO THE HOST CELL:
• WHICH PERMIT THEIR BACTERIAL HOSTS TO SURVIVE BETTER IN AN ADVERSE
ENVIRONMENT
• TO COMPETE BETTER WITH OTHER MICROORGANISMS OCCUPYING THE SAME
ECOLOGICAL NICHE.
• PLASMIDS THAT ENCODE FOR ANTIBIOTIC RESISTANT PROTEINS AND
MECHANISMS RESULT IN ANTIBIOTIC RESISTANCES IN BACTERIA POPULATION.
PLASMIDS : PROPERTIES
PLASMIDS PROPERTIES AND
AND CHARACTERISTICS
CHARACTERISTICS
• THE SIZES OF MOST PLASMIDS ARE RELATIVELY SMALL.
• GENERALLY IN SIZES OF ABOUT 1 KILOBASE TO OVER 300 KB, (LESS THAN ONE-
TWENTIETH THE SIZE OF THE CHROMOSOME.
• PLASMIDS OFTEN ONLY TAKE A SMALL PART OF THE CELL GENOME.
• BUT SOMETIMES THEY CAN BE LARGER THAN THE CHROMOSOME ITSELF (E.G. IN
VIBRIO CHOLERAE).
• PLASMIDS ARE WITH EITHER A "LOW" OR "HIGH" COPY NUMBER.
• IF PLASMID IS MAINTAINED WITH AN AVERAGE COPY NUMBER OF 10-30
PLASMIDS/CELL. THIS IS CONSIDERED LOW COPY NUMBER.
• IF THE PLASMID IS MAINTAINED WITH AN AVERAGE COPY NUMBER OF 100-200
PLASMIDS/CELL. THIS IS CONSIDERED A HIGH COPY NUMBER TYPE OF PLASMID.
• DIFFERENT PLASMIDS CAN CONSTITUTE EVEN UP TO 50% OF BACTERIAL
GENOME (E.G. IN BORRELIA BURGDORFERI).
PLASMIDS : TYPES
PLASMIDS TYPES OF
OF PLASMIDS
PLASMIDS
1. F PLASMID (FERTILITY PLASMID)
• THIS TYPE OF PLASMID CONFER AND
PROMOTE THE ABILITY TO TRANSFER
CHROMOSOMAL GENES (GENES NOT CARRIED
ON THE PLASMID) FROM A CELL CONTAINING
AN F PLASMID.
2. VIRULENCE PLASMID
• VIRULENCE PLASMIDS CONFER PATHOGENICITY. THEY ALSO CONFER THE
ABILITIES TO MORE SUCCESSFULLY COLONIZE A HOST ORGANISM SUCH AS
ANIMALS OR HUMAN BEING.
3. DEGRATIVE PLASMID
• DEGRATIVE PLASMIDS CONFER ON THE HOST BACTERIUM THE ABILITY TO
DEGRADE SOME UNUSUAL SUBSTANCES NOT OFTEN ENCOUNTERED IN THE
ENVIRONMENT. AN EXAMPLE IS THE TOL PLASMID IN PSEUDOMONAS PUDITA TO
FIX NITROGEN.
4. R PLASMID (RESISTANCE PLASMID)
• RESISTANCE PLASMIDS ARE PLASMIDS THAT ASSIST THE BACTERIUM THE
ABILITY TO GROW OR SURVIVE IN THE PRESENCE OF ANTIBIOTICS OR
ANTIMICROBIAL AGENTS (I.E. PENICILLIN, ERYTHOMYCIN), COLICINS OR SOME
METAL ION (I.E. MERCURY, NICKEL, AND ZINC). TRANSPOSONS
TRANSPOSONS
TRANSPOSONS
• TRANSPOSON ARE GENETIC MATERIAL THAT
CARRY ADDITIONAL PIECES OF DNA.
• TRANSPOSONS ARE PART OF OTHER GENETIC
ELEMENTS - CHROMOSOME, PLASMID.
• TRANSPOSONS ARE SEGMENTS OF DNA THAT
CAN MOVE AROUND TO DIFFERENT POSITIONS
IN THE GENOME OF A SINGLE CELL.
• FROM ONE SITE IN DNA TO ANOTHER AND
PLASMIDS OR BETWEEN DIFFERENT PLASMIDS
BUT WITHIN THE SAME CELL.
• THESE MOBILE SEGMENTS OF DNA ARE
SOMETIMES KNOWN AS "JUMPING GENES", R
FACTORS, "JUNK" DNA AND "SELFISH" DNA.
• "JUNK" BECAUSE THERE IS NO OBVIOUS
BENEFIT TO THEIR HOST.
• "SELFISH" BECAUSE THEIR ONLY FUNCTION SEEMS TO MAKE MORE COPIES OF
THEMSELVES.
• TRANSPOSONS OFTEN CARRY GENES ENCODING ENZYMES THAT INACTIVATE
ANTIBIOTICS AND METABOLIC ACTIVITIES OF A GREAT DIVERSITY OF TYPES.
TRANSPOSONS
TRANSPOSONS
• AS THE TRANSPOSONS MOVE TO DIFFERENT POSITIONS, THEY CAUSE:
• INCREASE (OR DECREASE) THE AMOUNT OF DNA IN THE GENOME.
• CAUSE MUTATIONS
• DELETIONS, INSERTIONS, INVERSIONS, REARRANGEMENTS OF THE GENOME.
• HOW DO TRANSPOSONS CAUSE MUTATIONS?
• IF A TRANSPOSON INSERTS ITSELF INTO A FUNCTIONAL
GENE, IT WILL PROBABLY DAMAGE IT. INSERTION INTO
EXONS, INTRONS, AND EVEN INTO DNA FLANKING THE
GENES (WHICH MAY CONTAIN PROMOTERS AND
ENHANCERS) CAN DESTROY OR ALTER THE GENE'S
ACTIVITY.
• FAULTY REPAIR OF THE GAP LEFT AT THE OLD SITE (IN
CUT AND PASTE TRANSPOSITION) CAN LEAD TO
MUTATION THERE.
• THE CAUSE OF THE MUTATIONS RESPONSIBLE FOR
SOME CASES OF HUMAN GENETIC DISEASES,
INCLUDING HEMOPHILIA.
• A TRANSPOSABLE ELEMENT IS THE BASIS FOR MOST
CURRENT GENETIC ALTERATION OF PLANTS.
TRANSPOSONS
TRANSPOSONS
• TRANSPOSONS CAN BE ON VIRUSES OR PLASMIDS WHICH CAN
INTRODUCE THEM INTO OTHER CELLS OR ON CHROMOSOMES.
• THERE ARE THREE DISTINCT TYPES:
• CLASS I TRANSPOSONS.
• ALSO KNOWN AS RETROTRANPOSONS.
• RETROTRANSPOSONS MOVE BY A "COPY AND PASTE" MECHANISM.
• THE COPY IS MADE OF RNA, NOT DNA.
• ABOUT 40% OF THE ENTIRE HUMAN GENOME CONSISTS OF
RETROTRANSPOSONS.
• CLASS II TRANSPOSONS - CONSISTING ONLY OF DNA THAT MOVES DIRECTLY
FROM PLACE TO PLACE.
• CLASS III TRANSPOSONS - ALSO KNOWN AS MINIATURE INVERTED-REPEATS
TRANSPOSABLE ELEMENTS OR MITES.
TRANSPOSONS
TRANSPOSONS
• TRANSPOSONS IN BACTERIA
• SOME TRANSPOSONS IN BACTERIA CARRY GENES FOR ONE OR MORE (USUALLY
MORE) PROTEINS IMPARTING RESISTANCE TO ANTIBIOTICS.
• WHEN SUCH A TRANSPOSON IS INCORPORATED IN A PLASMID, IT CAN LEAVE THE
HOST CELL AND MOVE TO ANOTHER.
• THIS IS THE WAY THAT THE ALARMING PHENOMENON OF MULTIDRUG ANTIBIOTIC
RESISTANCE SPREADS SO RAPIDLY.
• TRANSPOSITION IN THESE CASES OCCURS BY A "COPY AND PASTE MECHANISM.
THE ORIGINAL TRANSPOSON REMAINS AT THE ORIGINAL SITE WHILE ITS COPY IS
INSERTED AT A NEW SITE.
• TRANSPOSON INSERTIONS CAN BE ISOLATED AT A LARGE NUMBER OF SITES ON
THE BACTERIAL CHROMOSOME.
PROKARYOTIC RECOMBINANT
PROKARYOTIC RECOMBINANT DNA
DNA MECHANISMS
MECHANISMS
• RECOMBINANT DNA IS THE FUSION OF GENETIC ELEMENTS FROM
SEPARATE INDIVIDUALS/ORIGINS.
• THERE ARE 3 WAYS OF TRANSFERRING DNA INTO BACTERIAL CELLS:
• TRANSFORMATION
• BACTERIA GETTING GENES FROM NAKED DNA
• TRANSDUCTION
• PHAGE TRANSPORTING OF GENES
• CONJUGATION
• BACTERIA SWAP GENES THROUGH SEX PILI
PROKARYOTIC RECOMBINANT
PROKARYOTIC RECOMBINANT DNA
DNA :: TRANSFORMATION
TRANSFORMATION
• TRANSFORMATION OCCURS WHEN BACTERIA TAKE UP NAKED DNA FROM
THEIR SURROUNDINGS.
• DNA FROM A DONOR BACTERIAL CELL (RELEASED AFTER ITS DEMISE) IS TAKEN
UP BY ANOTHER BACTERIAL CELL (RECIPIENT, NOT NECESSARILY THE SAME
SPECIES).
• SOME BACTERIA HAVE SPECIALIZED MEMBRANE PROTEINS TO BRING DNA INTO
THEIR CELLS (E.G. BACILLUS SPP.)
• MANY BACTERIAL HOWEVER REQUIRES CA2+ TO STIMULATE THE UPTAKE
OF FOREIGN DNA.
• THIS IS THE PRINCIPLE BEHIND THE TRANSFORMATION OF BACTERIAL IN
EXPERIMENTS.
• UP TO 10 KB OF DNA CAN BE TRANSFERRED IN THIS FASHION.
PROKARYOTIC RECOMBINANT
PROKARYOTIC RECOMBINANT DNA
DNA :: TRANSDUCTION
TRANSDUCTION
• TRANSDUCTION IS THE MOVEMENT OF DNA FROM ONE BACTERIUM TO
ANOTHER BY BACTERIOPHAGES.
• GENERALIZED TRANSDUCTION CAN OCCUR FOR ANY GENE.
• TRANSFER OF INFORMATION FROM ONE HOST TO ANOTHER (BOTH MUST
BE SUSCEPTIBLE TO THIS PHAGE) THAT OCCURS TWO WAYS
• GENERALIZED
• CHUNKS OF HOST DNA ARE ACCIDENTALLY PACKAGED INTO CAPSIDS BY
MISTAKE.
• SPECIALIZED:
• BACTERIOPHAGE INSERT INTO BACTERIAL CHROMOSOME (PROVIRUS STATE)
• WHEN VIRUS LEAVES THE PROVIRUS STATE IT MAY TAKE SOME BACTERIAL
GENES CLOSE TO THE INSERTION SITEWITH IT
• THE AMOUNT OF DNA THAT CAN BE PACKAGED INTO A PHAGE PARTICLE
IS LESS THAN 100 KB.
PROKARYOTIC RECOMBINANT
PROKARYOTIC RECOMBINANT DNA
DNA :: TRANSDUCTION
TRANSDUCTION

CONJUGATION
PROKARYOTIC RECOMBINANT
PROKARYOTIC RECOMBINANT DNA
DNA :: CONJUGATION
CONJUGATION
• CONJUGATION IS THE DIRECT TRANSFER OF
GENES BETWEEN JOINED BACTERIA.
• IT IS A FORM OF BACTERIAL "SEX"
• USUALLY ONLY A FEW GENES TRANSFERRED.
• HOWEVER, AN ENTIRE CHROMOSOME CAN BE
TRANSFERRED THIS WAY.
• CONJUGATION IS SPECIFIC, UNI-DIRECTIONAL
TRANSFER OF CONJUGATIVE PLASMIDS FROM ONE
BACTERIUM TO ANOTHER.
• DONOR DOES NOT LOSE DNA; HIS CHROMOSOME
IS DUPLICATING WHILE HE TRANSFERS GENES.
• THE RECIPIENT IS CALLED TRANSCONJUGATE.
• TRANSFER OF GENES IS SLOW; CAN BE USED TO MAP BACTERIAL GENES.
PROKARYOTIC RECOMBINANT
PROKARYOTIC RECOMBINANT DNA
DNA :: CONJUGATION
CONJUGATION
• DURING CONJUGATION, THE MECHANISMS INVOLVED ARE :
1. "MALE" DONOR BACTERIUM PROJECTS
A HOLLOW TUBULES CALLED PILI:
• F PLASMID HAS GENES NEEDED TO MAKE
THE SEX PILI.
• MALE BACTERIUM HAS AN F PLASMID.
• DNA TRAVELS IN ONLY ONE DIRECTION.
2. PILI CONNECT TO A "FEMALE" RECEPTOR :
• "FEMALE" BACTERIUM HAS NO F PLASMID.
• "FEMALE" CAN TAKE UP AN F PLASMID
AND BECOME "MALE" .
• RECEPTOR FEMALE BECOMES PARTIALLY
DIPLOID.
OVERVIEW OF
OVERVIEW OF PROKARYOTIC
PROKARYOTIC RECOMBINANT
RECOMBINANT DNA
DNA MECHANISMS
MECHANISMS