Genetic recombination

MOLECULAR BIOLOGY

1. GENETIC RECOMBINATION IN BACTERIA

2. INTRODUCTION:- Bacteria do not reproduce sexually like eukaryotic organisms because their requirement of sexuality are met through certain alternate pathway of genetic recombination which are called “CONJUGATION”,”TRANSFORMATION”& “TRANSDUCTION”. These 3 processes mediate transfer of genetic material (DNA) from one bacterial cell (Donor)to the other (recipient)making possible the subsequent recombination events.

3. The most obvious difference between these 3 processes is the mode of transfer of genetic material (DNA) from donor to recipient cell:- In “CONJUGATION” donor cell transfers its DNA to recipient cell only when both the cells are in physical contact through a specialized sex-pilus or conjugation tube. In “TRANSFORMATION” there is transfer to a cell-free or “naked” DNA from donor cell to recipient cell. In “TRANSDUCTION” the transfer of genetic material (DNA) from donor cell to recipient cell is mediated by a bacteriophage.

4. THE RECOMBINATION IN BACTERIA USUALLY TAKES PLACE FORMING “ PARTIAL NORMAL DIPLOIDS” also called “heterozygotes” or “ merozygotes”.THUS , the recipient cells are converted to heterozygotes or merozygotes contain fragments of donor DNA “the exogenote ” ans a complete recipient dna “”the endogenote”.

5. 1}conjugation Lederberg and tatum discovered conjugation in “E.coli” in the year 1946 and detailed studies were made by Woolman and Jacobin 1956. “conjugation is a process by which genetic material is transferred from one bacterial cell (Donor cell or male cell) to another (recipient cell or female cell) through a specialized intercellular connection called “sex pilus or conjugation tube”. The maleness and femaleness of bacterial cells are determined by the presence or absence of “F-plasmid “(also called F-factor or sexfactor ).

6. F-plasmid an extrachromosomal genetic material is always present in the cytoplasm of donor or male cells and the , latter develop specialized cell surface appendages called “F- pilli or sex pilli “under the control of F-plasmid recipient or female cells always lack F-plasmids and therefore , F- pilli are not present on their surface F-plasmid or F-factor can exist in 2 different states:-

7. AUTONOMOUS STATE INTEGRATED STATE It lies free in the cytoplasm and this state is integrated into the replicate independent of the bacterial bacterial chromosome(DNA) and replicate alongwith it. Chromosome (DNA) A donor or male cell containing F-factor in autonomous a donor or male cell containing F-factor in integrated state is called “ F+cell ” state is called”Hfr cells” or high frequency male cells. however, the recipient or female cell lacks F-factor and this is called “F – cells “.

8. 1}conjugation between a F+(Donor)cell and a f- (recipient) cell:- In conjugation between a F+(donor) cell and F-(recipient) cell, it autonomous F- factor (F-plasmid)which is transferred never the bacterial DNA. When the 2 cells (F+&f-) come close to each other , the F- pilus of the F+(Donor) cell attaches with the F- (recipient)cell and acts as a conjugation tube. Simultaneously , the double –stranded circular F-factor DNA is nicked at a specific point and begins to replicate producing a single –stranded copy of the F-factor DNA which migrates through the tube into the cytoplasm of the F-(recipient )cell. It becomes double stranded and circulars and lies free in the cytoplasm thus , rendering the recipient cell to become F+ donor cell. In this way , mixing a population of F+ (donor)cells with a population of F+ (recipient )cells results in the conversion of virtually all the cells in the population becoming F+ (donor) cells.

9. Conjugation between Hfr donor cells and recipient (f-) cell:- The Hfr donor cells are considered to be fertile because unlike F+(donor) cells , their chromosomal segments are transferred from donor to recipient cells and the F- factor remains in sites. When 2 cells ( Hfr and F-) come in contact , a conjugation tube develops between them . The circular DNA of Hfr donor cell is nicked and replication is initiated, The integrate F-factor always lies at the rear end of the DNA molecule. The replication of DNA starts towards the end near the conjugation tube & newly synthesized single strand starts migrating through the tube into the recipient (F-) cell. In nature the mating of 2 cells exists for a short period and gets interrupted resulting in the migration of only a portion of the donor DNA into the recipient cell. The genes of the newly entered DNA fragment may replace the homologous genes of the DNA of the 43recipient cell , resulting in a recombination genetic material . The newly formed recombinant genetic material now possess those male charactters that hav ebeen transferred through recombination with the migrated DNA fragment .

10. conjugation BETWEEN F’(F PRIME) MALE AND F-(RECIPIENT)CELL(SEX-DUCTION):- Existence of Hfr donor cells is not absolute and the F-factor integrated into the bacterial DNA of Hfr donor cells may dissociate and become free in the cytoplasm . The dissociation may be occasionally anomalous during which the dissociated F-factor may bring with it some genes of the bacterial chromosomes.’ When F-prime male conjugates with F- (recipient cell) the F-factor is transferred from donor to the recipient cell and such a recipient bacterial cell becomes heterozygous for the part of the bacterial chromosome which the F-prime factor had obtained during its anomalous dissociation. Transfer of F-prime factor to recipient cell apparently occurs by the same mechanisms as F- factor transfers in Hfr and F- cell mating . Genetic recombination of this type mediated by F PRIME FACTOR is called “SEX-DUCTION OR F0-DUCTION”.

11. 2 }transformation Transformation is a process of genetic recombination was first studied by “GRIFFITH” in 1928 (an English bacteriologist ). He took 2 strains of the bacterium Streptococcus pneumoniae. One of the 2 strains was virulent or pathogenic and capsulated normal which formed smooth colonies on culture medium . and The other strain was non-pathogenic or avirulent and non-capsulated which formed rough colonies on culture medium.

12. He experimented on mice as summarised below:-

13. It is obvious from Griffiths experiment that the avirulent or non-pathogenic strain becomes virulent or pathogenic when mixed with heat –killed (dead) virulent strain ,thus , causing the death of mice. Griffith named this change of avirulent into virulent strains as “Transformation”. Griffith reasoned that there was a transfer of some factor from heat-killed (dead) virulent strain to the virulent strain and called it “TRANSFORMING PRINCIPLE”. He said that the transforming principle was the polysaccharide of capsule of heat –killed virulent strains.

14. The idea of polysaccharide as transforming principle came to an end in 1944 when AVERY MACLOED and MCCARTY showed that it is the DNA which works as “TRANSFORMING PRINCIPLE “ not the polysaccharide capsule .They proved for the first time that DNA is the genetic material in organisms. In transformation a free naked DNA molecule is transferred from a donor to recipient bacterial cell. The donor bacterium undergoes lysis to free the DNA molecule and the recipient bacterium must be component to receive it . The competence of bacterial cell is not a permanent feature it has been demonstrated in relatively few bacterial genera and depends upon the growth of bacteria and the environmental conditions.

15. When a donor DNA comes into contact with the competent bacterial cell , it first binds on the cell surface and then is taken up inside the cell. In some cases it is observed that the double strand DNA enters inside the bacterial cell as such and its one strand is degraded by “ENDONUCLEASE” enzyme there in leaving single – strand DNA whereas In others species of Bacillus and Streptococcus it appears that only single strand DNA enters the recipient bacterial cell.

16. An endonuclease enzyme degrades one of the strands of double stranded DNA of recipient bacterial chromosome in corresponding region and this gap is filled by the donor single stranded DNA with the help of ligase enzyme which joins it with the DNA of the recipient bacterial chromosome. If allelic forms of donor and recipient genes are not identical the donor DNA forms a “HETERODUPLEX” with the recipient bacterial DNA. When the bacterial cell containing “HETERODUPLEX” it undergoes binary fission the hetroduplex replicates forming 2 “HOMODUPLEXES”.

17. One of these is a normal duplex which is all recipient in origin and the daughter cell containing it is like the recipient bacterial cell . and The other homoduplex is a transformed duplex (hybrid genome) different from that of either the donor or the recipient bacterial genome . The daughter cell containing transformed duplex is “TRANSFORMED CELL” and contains some of the characteristics of the donor bacterial cell which are inherited progeny to progeny .

18. 3}transduction:- This process of genetic recombination was discovered by “ZINDERAND LEDERBERG”(1952) in Salmonella typhimurium during their experiments with the objective of discovering whether E.coli type of genetic exchange also existed in Salmonella typhimurium. In transformation where in DNA (naked) is transferred or fragments of DNA are transferred from one bacterial cell to the other but During transduction phage mediated process of genetic material transfer in bacteria.

19. The bacteriophage acquires a portion of the bacterial DNA of the bacterial DNA the host cell in which it reproduces and then , transfers this acquired DNA to another bacterial cell to which it infects such bacteriophage is called “TRANSDUCING BACTERIOPHAGE”. Transduction is of 2 types:- i )Generalized transduction {non-specialised} ii)Specialized transduction {restricted }

20. A}generalized transduction :- Transduction which results in transfer of any bacterial gene from one bacterial cell to the other is reffered to as “GENERALIZED OR NON-SPECIALIZED TRANSDUCTION”. It is mediated by some virulent phages and certain temperate phages like E.coli phage P1 , Salmonella phage P22 and Bacillus subtilis phages PBS1 and SP10 are such phages . In generalized transduction some of the developing progeny phages during their normal lytic cycle may accidentally acquire pieces of bacterial DNA. Such phages after the lysis of the host bacterial cell and their release attach to and inject their DNA into a new recipient cell but fail to re-establish lytic cycle. Once inside the recipient bacterial cell the injected DNA may be degraded by NUCLEASES in which genetic exchange does not occur. The injected DNA may undergo integration resulting in homologous recombination. As a result , the transduced cell may possess new combination of genes .Now , the transduced bacterial cell now undergoes,usually binary fission and produces progeny cells containing new combination of genes.

21. B}specialized transduction:- Specialized transduction which leads to the transfer of only specific or restricted genes from donor to recipient cell. It is mediated by temperate bacteriophage {Lambda phage, mu phage and Ø 80 phage} that usually incorporate (integrate)their DNA into the bacterial chromosome. The phage DNA is called ”PROPHAGE” in its integrated state with the bacterial chromosome , the bacterium having a prophage is said to be “lysogenic “ and the phage –host –relationship is called “LYSOGENY”.

22. During transition the prophage is usually excised precisely from the specific site of integration in its exactly originally form but It may excise imprecisely so that it takes specific portion of bacterial chromosome which lies close to the site of prophage insertion and leaves a portion of its own DNA remaining integrated within the bacterial chromosome . such prophage is called “SPECIALIZED TRANSDUCING PRINCIPLE” and is packed into developing phage particle inside the host bacterial cell. Phage particle so developed is called “specialized transducing phage”and is released after the host bacterial cell undergoes lysis.

23. When a viable specialized transducing phage infects a new bacterial cell its specialized transducing principle that already contains specific portion of bacterial chromosome inserts into the recipient bacterial chromosome. Since , the specialized transducing phage is “DEFECTIVE” phage as it has lost some genes during excision and its functions in recipient bacterial cell only when the latter is already infected by another phage , i ,.e , it contains missing genes and there by , complements the lost phage functions of specialized transducing phage. The partial diploid contains 2 copies of the concerned genes, one from donor bacterium and other recipient bacterium and is unstable. AS a result bacterial cells containing gene of donor bacterium and these containing gene of recipient bacterium segregate at a frequency of about one in 1000 cell division.

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