Docstoc

Recombination_ Transformation

Document Sample
Recombination_ Transformation Powered By Docstoc
					                     1




MICROBIAL GENETICS
                                                                          2

                        MICROBIAL GENETICS
RECOMBINATION
 Homologous, Site-specific, illegitimate
 Breaking and Re-joining
 Parental chromosomes, Recombinant chromosomes

TRANSFORMATION

 Streptococcus pneumoniae:
    Wild-type, smooth colonies, capsule production, pathogenic
    Rough mutant, no capsule production, non-pathogenic

 Transformation of auxotrophic mutants
   Donor cells, donor DNA, recipient cells, recombinants, transformants
   Phenotypes, Selection

 Transformation by plasmid DNA
   Antibiotic resistance plasmids
   Penicillin-resistance, penicillinase, beta-lactamase
                                INTRODUCTION TO MICROBIAL GENETICS                                                                  3
       Genetic information is very stable (in most organisms) because nature has evolved mechanisms to repair damaged
DNA and to ensure great fidelity in DNA replication, As a result, mutations occur very randomly. That is very good for each
species because they continue to survive and compete successfully in their environment. On the other hand, new kinds of
organisms are clearly needed from time to time, for example to take advantage of changing environments, either good or bad
changes in the environment. There are two ways that new organisms can arise.
       Mutation. Some times, mutants are improved organisms and can grow and survive and compete better than the
original organism. An example is ability to grow in the presence of some antibiotic. Other times, mutations will create defects
and the mutants which suffer those mutations will not be able to survive.
       Genetic Exchanges. The second way is by combining genes from different organisms to create new organisms.
These exchanges move genes or larger fragments of chromosomes from one organism to another and create recombinant
organisms with combinations of properties from the original organisms.
A. NATURAL EXCHANGES OF CHROMOSOMES OR CHROMOSOME FRAGMENTS IN MICROBES. Genetic exchanges
occur in microbes by three natural mechanisms; Transformation, Transduction, and Conjugation.
       Transformation involves the breakage of lysis of one cell (the donor) with the resulting breakage of the chromosome
and release of chromosomal fragments into the environment, the uptake of those chromosomal fragments another cell (the
recipient) and the incorporation of donor DNA fragment into the chromosome of the recipient.
       Transduction is the movement of chromosomal fragments from one cell (the donor) by growth of some phage on the
donor cell, the incorporation of donor DNA into the chromosome of the phage (or packaging of donor DNA into phage heads)
generating transducing phages or transducing particles, the subsequent infection of another cell (the recipient) by the
transducing phage or particle, injection of donor DNA into the recipient cell, and incorporation of donor DNA into the
chromosome of the recipient.
       Conjugation is the transfer of chromosome fragments (or entire chromosomes, but this is rare) from male cells
(donors) into the cytoplasm of female cells (recipients). The transferred fragments are incorporated into the chromosome of
the recipients.
B. BACTERIAL PLASMIDS Plasmids are small DNA molecules which are not normally part of the chromosome and which are
usually circular and which usually are not essential for the growth of the organism. They often code for only 10 to 50 genes.
Entire plasmids can be transferred from cell to cell by all three procedures, transformation, transduction and conjugation. It is
not essential that the transferred plasmids be incorporated into the chromosome of the recipient.
C. GENETIC RECOMBINATION Breaking and rejoining DNA fragments is sometimes involved in generating chromosome
fragments to be transferred and breaking and rejoining is always required to incorporate DNA fragments into the recipient
chromosome. This breaking and rejoining is called genetic recombination. Three types of recombination:
       Homologous recombination- Depends on breaking and rejoining DNA in regions of identical or very similar
nucleotide sequence. There is no requirement for any specific nucleotide sequence. That is, any regions of two DNA
molecules can be broken and rejoined, so long as the nucleotides in those two molecules have the same or very similar
sequences. For example, any region of the entire E.coli chromosome is subject to homologous recombination.
       Site-specific recombination- Depends on breaking and rejoining two molecules at region four sites, where defined
sequences of nucleotides pairs are recognized by an enzyme which catalyzes breaking and rejoining. For example, phage
lambda integrate into the E. coli chromosome at one specific, unique site, the prophage attachment site.
       Illegitimate recombination. Very rarely, breaking and rejoining can occur between two regions of no similarity of
nucleotide sequence. This happens by mistake. Although not common, it occurs and in huge populations, as in bacterial and
phage populations, it certainly can be found to occur.
D. RECOMBINANT DNA TECHNOLOGY. Chromosome fragments (genes) from one organism can be joined to plasmids of
virus DNA in the test tube and introduced into another, often unrelated organism to generate recombinant organisms.
                                              4
         GENETIC RECOMBINATION

BREAKING AND JOINING DNA
   CHROMOSOMES / MOLECULES / FRAGMENTS

1. HOMOLOGOUS-
     ANY REGIONS OF DNA AS LONG AS THEIR
     NUCLEOTIDE SEQUENCES ARE IDENTICAL OR
     VERY SIMILAR

2. SITE SPECIFIC-
      TWO FRAGMENTS ONLY IF THEY CONTAIN
      SPECIFIC SITES (NUCLEOTIDE SEQUENCES)
      RECOGNIZED BY SPECIFIC ENZYME

3. ILLEGITIMATE-
       FRAGMENTS WITH NO SIMILAR SEQUENCES
       RARE;
       ERRORS
                              5



 WAYS TO TRANSFER DNA:

     TRANSFORMATION

       TRANSDUCTION

       CONJUGATION

ALL DEPEND ON RECOMBINATION
   (WITH SOME EXCEPTIONS)
                                                                 6
             HOMOLOGOUS RECOMBINATION
       SIMILAR SEQUENCES BETWEEN TWO GENES
                 (OR WITHIN ONE GENE)
       a             b
5'                             3'
                                      MUTANT a      MUTANT b
3'                             5'

5'     A                   B   3'
                                     WILD-TYPE A   WILD-TYPE B
3'                             5'
 PARENTAL CHROMOSOMES                     [DEFINE ALLELE]

     NICK ONE STRAND
     GENERATE 3' END ON
       SINGLE-STRAND REGION
                                5'
     INVASION OF DUPLEX                  3'                 5'
        BY SINGLE-STRAND
     HYDROGEN BONDS-
       COMPLEMENTARY
       NUCLEOTIDES              5'
                                                            5'
                                                               7
    NICK LOWER STRAND, TOP DUPLEX
        TRANSFER TO LOWER DUPLEX
DNA LIGASE JOINS ADJACENT 5' AND 3' ENDS
                                    a                 b
                              5'
                                                          5'
                                           3'

                                    A                 B
                              5'
                                                          5'
                                           3'
                NICK REMAINING PARENTAL STRANDS
                ROTATE AND EXCHANGE RIGHT ENDS    +
          a                  B
   5'
                                   5'

                             b
          A
   5'
                                   5'
                                                                8
                LIGASE JOINS ADJACENT 5' AND 3'
                 RECOMBINANT CHROMOSOMES

5'   a                 B   3'
                                 MUTANT a         WILD-TYPE B
3'                         5'

5'
     A                 b   3'
                                WILD-TYPE A        MUTANT b
3'                         5'



         IN SUMMARY:
           PARENTAL CHROMOSOMES
                                       a      b
                                       A      B

           PRODUCTS OF RECOMBINATION
                                 a   B
                                 A   b
                                             9
  RESOLUTION BY NICKING HORIZONTAL
         a       b      c
 ONE
PARENT

         A       B      C
SECOND
PARENT




                     CUTTING AND LIGASE
         a       b      c

         a       B      c      REPLICATION
         A       B      C

         A       b      C
                                             10

                  [       = NEW SYNTHESIS]




a   b   c             A       B         C


a   b   c             A       B         C
        LIKE PARENTAL
    +                         +
a   B   c             A       b         C


a   B   c             a       b         c
        RECOMBINANT
        CHROMOSOMES
         SINGLE STRAND FRAGMENTS CAN                            11
         RECOMBINE INTO A CHROMOSOME

MUTATION =   5'                                 3'
             3'                                 5'

                        3'                 5' WILD-TYPE FRAGMENT
                  INVASION
                  5'                            NICK AND DISPLACE
                             3'              5'   ONE RECIPIENT
                                             5'      STRAND
                                                   FRAGMENT;
                                                      LIGASE

                                            +
                                                      DEGRADED
                         REPLICATION
                                       [    = NEW SYNTHESIS]




      MUTANT CHROMOSOME           WILD-TYPE CHROMOSOME
  TRANSFORMATION - LINEAR FRAGMENTS OF                    12
             CHROMOSOME
            STREPTOCOCCUS PNEUMONIAE

    COLONY                                RESULT OF
                        INJECT
  MORPHOLOGY                           INJECTING BUNNY

    SMOOTH,
                                          DISEASE -
MUCOID,GLISTENING
                       WILD-TYPE       BLOOD CULTURE -
    CAPSULE
                                          WILD-TYPE
(POLYSACCHARIDE)

                                         NO DISEASE -
ROUGH, NO CAPSULE       MUTANT
                                       CULTURE NEGATIVE


                      HEAT-KILLED        NO DISEASE -
  NO COLONIES
                       WILD-TYPE       CULTURE NEGATIVE

                          MIX-            DISEASE -
                    MUTANT AND HEAT    BLOOD CULTURE -
                    KILLED WILD-TYPE      WILD-TYPE
                                    13




          NEGATIVE STAIN
CELLS SURROUNDED BY LARGE CAPSULE
                                            14
       STREPTOCOCCUS PNEUMONIAE
             ROUGH MUTANT

= MUTATION IN
  CAPSULE
  BIOSYNTHESIS
  GENE                        DNA FROM
                              WILD-TYPE
                                CELL
                               WILD-TYPE
                               CAPSULE
                             BIOSYNTHESIS
                                 GENE




      HOMOLOGOUS
     RECOMBINATION
                                                     15



                                   FRAGMENT
                                   DEGRADED
                              [   = NEW SYNTHESIS]
             BINARY FISSION




                WILD-TYPE
ROUGH             CELL
MUTANT



         WILD-TYPE GENE
           EXPRESSED
                                                 16
STREPTOCOCCUS HEMOPHILUS BACILLUS

DONOR DNA: WILD-TYPE LEU+




                                 LEUCINE


RECIPIENT CELLS: LEUCINE REQUIRING MUTANT LEU-

     • EXTRACT DNA FROM DONOR CELLS
     • MIX DONOR DNA AND RECIPIENT CELLS
     • RECIPIENT CELLS TAKE UP DONOR DNA
     • HOMOLOGOUS RECOMBINATION
     • SELECT WILD-TYPE RECOMBINANTS
       (TRANSFORMANTS)
                                                  17
     SELECTING     LEUCINE+ TRANSFORMANTS


DONOR DNA (LEU+)                GLUCOSE MEDIUM
    2-3 µg                        [NO LEUCINE]




RECIPIENT CELLS
 (LEU- MUTANT)
    ~1 x 108




                                      LEU+
DONOR DNA AND
                                TRANSFORMANTS
RECIPIENT CELLS
                                   (~102 - 103)
                                            18
TRANSFORMATION BY PLASMID DNA


                            PLASMID


            PENICILLINASE
                              GENE FOR
                            PENICILLINASE


 BACTERIUM RESISTANT TO PENICILLIN
     ISOLATION OF PLASMID DNA
           •BREAK CELLS
   •SEPARATE PLASMID DNA FROM
     CHROMOSOME FRAGMENTS
       •PREP OF PLASMID DNA
            TRANSFORMATION BY PLASMID DNA                        19
                                   RICH MEDIUM
                    RICH MEDIUM
                                  AND PENICILLIN

• CONTROL - PLATE
  PLASMID DNA
     ~1 - 10µg
   [PENICILLIN
  RESISTANCE]



• CONTROL - PLATE
   PENICILLIN-
SENSITIVE CULTURE
   ~108 CELLS


 • MIX – PLASMID
  DNA (1 - 10µg)
       AND
   • PENICILLIN-                                   PENICILLIN-
SENSITIVE CULTURE
                                                   RESISTANT
    [RECIPIENT]
      GENERATING A SS REGION WITH A 3’ END-
           REC BCD ENZYME + CHI SITE

                                                C
                                          B     D
CHROMOSOME FRAGMENT
(RESULT OF IONIZING RADIATION;       A DNA HELICASE WITH
OTHER DAMAGE)                        SS NUCLEASE ACTIVITY
                                     (3’ 5’ AND 5’ 3’)

1. UNWINDS DS ~1000 BP/SEC
   CLEAVES 3’ END (PREFERENTIALLY)




                                          SSB
2. ENCOUNTERS  SITE
                                   = 5’ GCTGGTGG 3’
                                  CHANGES REC BCD
                                    SPECIFICITY




3. REC BCD 5’- 3’ NUCLEASE HYDROLYZES 5’ END
   (PREFERENTIALLY)
4. GENERATES A 3’ END WITH  SITE
   LOADS REC A PROTEIN WHICH COATS SS
   AND CATALYZES HOMOLOGOUS PAIRING
   AND STRAND EXCHANGE

                               


                                    REC A PROTEIN

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:2
posted:5/30/2012
language:Latin
pages:22