Recombination model and cytological basis of crossing over

Genetics

1. RECOMBINATION MODELS & CYTOLOGICAL BASIS OF CROSSING OVER Term paper presentation GP-502 SUBMITTED TO: Dr. D. Shivani PROFESSOR DEPT. OF GENETICS AND PLANT BREEDING SUBMITTED BY: ANIL KUMAR RAM/2020-68 GPBR

2. CONTENTS Introduction Types of recombination Homologous or generalized recombination Site-specific recombination Transposition Models for homologous recombination Models involving single strand breaks: Holliday model Models involving double strand breaks Cytological basis of crossing over Experiment of stern in drosophila Studies Creighton and McClintock in Maize Case study

3. introduction Recombination may be described as production of new combination of linked genes. It occurs between precisely homologous sequences in such a manner that not a single base pair is added to or lost from recombinant chromosomes. Recombination involves physical exchange of material between duplex DNAs.

4. Three different types of recombination Homologous or generalized recombination Site-specific recombination Transposition

5. Homologous or generalized recombination Homologous recombination involves the exchange of precisely corresponding sequences between homologous DNA duplexes. In eukaryotes, it occurs during the four strand stage of meiosis, involves only two of the four chromatids of a bivalent. It may occur at a lower rate in heterogametic sex.

6. Site-specific recombination Site-specific recombination occurs between specific pairs of DNA sequences since the enzyme involved can act only on a particular pairs of target sequences. This types of recombination has been characterized in prokaryotes, e.g., integration of phage genome into bacterial chromosome.

7. Transposition In transposition, breakage and reunion of DNA strand allows one DNA sequence to be inserted into another without reliance on sequence homology. The movable DNA segments called transposons.

8. Models for homologous recombination Models involving single strand breaks: Holliday model Meselson & Raddling Model Models involving double strand breaks: Szostak-Orr-Weaver- Rothestein -Stahl model

9. Holliday model Proposed by Robin Holliday in 1964. According to this model, 1 st an endonuclease produce single strand nicks at identical points in the two homologous parental DNA molecule in the strands having same polarity. Segment of single strands on one side of each nick are then displaced from their complementary strands up to some distance. Displaced single strand then crossover and pair with intact complementary strand of homologous chromosomes producing a joint molecule.

10. Cont… Each duplex in a joint molecule has a region in which it has one strand from each of the two parental DNA duplexes, this region is called hybrid DNA or heteroduplex DNA. The two single strand nicks remaining in the two duplexes are then joined by DNA ligase. The joint molecule undergoes reorientation to form an x-shaped structure called chi forms. One end of chi form now rotates by 180 ⁰ to form Holliday structure. Holliday structure is resolved by enzyme catalysed breakage and re-joining of complementary DNA strands to produce two recombinant DNA molecule.

11. Cont… An endonuclease now induce single strand nicks (in those strands that were not nicked brfore) in the holliday junction. As a result two splice recombinant DNA duplexes will be generated. The recombinant duplexes will have one nick each, which is then sealed by DNA ligase.

12. Models involving double strand breaks In 1983, J. Szostak and colleagues put forth a different model, initiated by double-strand break in one of the double helices. The DNA duplex in which the break is induced is called recipient duplex. The break is induced by endonuclease followed by 5’ →3 ‘ exonuclease activity to widen the gaps formed in the double helix and create 3 single-stranded tails on both sides of the break. One of the single stranded 3 ‘-end now invades the homologous region of other DNA duplex, called donor duplex.

13. Homologous strand in the donor duplex is replaced by invading strand. The displaced strand of donor duplex is now pair with other single stranded 3 ‘- end of recipient duplex. Repair synthesis of previously digested DNA using 3 ‘-end as a primer and strand of donor duplex as template fills the gap. Nicks remaining in two duplexes are sealed by DNA ligase. Cont…

14. Branch migration converts the two DNA duplexes into a joint DNA molecule having two recombinant joints. The two joints are resolved in opposite ways to produce a recombination event. Cont…

15. CYTOLOGICAL BASIS OF crossing over Experimental evidence of crossing over was provided in 1931 independently by:- Curt Stern in Drosophila & Creighton and McClintock in Maize

16. Experiment of stern in drosophila In his experiment, Stern used a drosophila female in which one X chromosome was shorter than normal. This chromosome had a recessive gene car (carnation eye colour) and dominant gene B (Bar eye shape). The other X chromosome of this female was of normal length, but a segment of Y chromosome was translocated into short arm. This chromosome had the dominant gene car + (wild type allele of car, produces red eye colour) and recessive gene B + (wild type allele of B, produces ovate eye shape).

17. Stern test crossed this female to a car B + (carnation normal) male. As expected the following four types of flies were recovered in test cross progeny: Red, normal (car + B + )---Parental type Red, bar (car + B)---Recombinant type Carnation, normal (car B + )---Recombinant type Carnation, bar (car B)---Parental type (Allele contributed by the test cross parent are not shown) Cont…

18. If crossing over involves exchange of sister chromatid between homologous chromosome then one X chromosome of recombinant individual would be the product of such exchange. Therefore Carnation, normal (car B + ) flies are expected to have a normal X chromosome without the attached Y chromosome. Red, bar (car + B) will have one short X chromosome with attached Y segment. Cont…

19. Stern observed very close correspondence between expectation and results actually obtained. He concluded that: During meiosis, there is exchange of precisely homologous chromatin segment between homologous chromosome. Crossing over is responsible for recombination between linked genes. Cont…

20. Studies Creighton and McClintock in Maize A similar conclusion was reached by Creighton and McClintock from their study in Maize. They used a maize plant in which one chromosome 9 was normal and had recessive gene c (colourless aleurone) and dominant gene Wx (nonwaxy endosperm). The chromosome 9 of this plant had a knob and was involved in unequal reciprocal translocation with chromosome 8. This chromosome had the dominant gene C (coloured aleurone) and the recessive gene wx (waxy endosperm).

21. This plant was testcrossed with the double recessive strain (c wx/c wx) having normal chromosome. The phenotype as well as morphology of chromosome 9 of test cross progeny were recorded. The data were in prefect agreement with the expectation as was the case in Drosophila experiment. Cont…

22. CASE STUDY

23. Name of Journal: BMC Plant Biology NAAS Score: 9.67 Received: 18 November 2019 Accepted: 8 July 2020 Published online: 16 July 2020

24. Background Current excitement about the opportunities for gene editing in plants have been prompted by advances in CRISPR/Cas and TALEN technologies. CRISPR/Cas is widely used to knock-out or modify genes by inducing targeted double-strand breaks (DSBs) which are repaired predominantly by error-prone non-homologous end-joining or microhomology-mediated end joining. Gene replacement (or gene targeting) by homology directed repair occurs at extremely low frequency in higher plants making screening for useful events unfeasible.

25. Homology directed repair might be increased by inhibiting non-homologous end-joining and/or stimulating homologous recombination (HR). Here we pave the way to increasing gene replacement efficiency by evaluating the effect of expression of multiple heterologous recombinases on intrachromosomal homologous recombination (ICR) in Nicotiana tabacum plants. Cont…

26. METHODS Bacterial and human recombinases cloning:- The coding sequences of bacterial recombinases ( RecA , RecG , RuvC ) and human recombinases (Rad51, Rad52 DMC1) were amplified by polymerase chain reaction (PCR) . The PCR product were cloned in pGEM ®-T Easy vector (Promega). In vitro expression:- Polyprotein constructs were used with wheat germ transcription– translation system (TNT®, Promega) in the presence of [35S]-Methionine. Radiolabelled protein products were separated in 10% SDS–PAGE and detected by autoradiography. Plant expression vector pGSC :- All bacterial and human recombinases constructs in pGEM ®-T Easy were transferred into pGSC plasmid using convenient restriction enzymes.

27. a. The coding sequences of bacterial ( RecA , RecG and RuvC ) and human (Rad51, Rad52 and DMC1) Recombinases. b. The transgene used as ICR substrate in the tobacco transgenic line N1DC4 is formed of two defective overlapping fragments of β -glucuronidase (GUS)

28. SDS-PAGE of in vitro TNT products

29. Plant transformation:- The constructs in pGSC binary vector were transferred into Agrobacterium tumefaciens strain LBA4404 . Agrobacterium clones were used to transform tobacco seedlings of N1DC4 line. The transgenic lines were selected and transferred to the glasshouse. At maturity pollen and seeds of the obtained primary transformants were collected. Transgene segregation and seed scoring:- T1 seeds were plated on MS medium in the presence of 100 mg/l of sulphonamide and scored for resistance. The lines showing 3:1 segregation ratio were selected to make homozygotes. H omozygous lines and N1DC4 control were grown in the glasshouse and their pollen and seeds were collected. The seeds of ten dry pods per plant were pooled and their number estimated by weight method. Cont…

30. Intrachromosomal recombination (ICR) assay:- N1DC4 is a homozygous line containing β- glucuronidase (GUS) based transgene as a substrate for Intrachromosomal recombination (ICR). The transgene is formed of two defective overlapping GUS fragments in direct orientation and separated by hygromycin resistance gene ( hpt ). ICR restores a functional GUS gene that can be detected by histochemical staining as blue spots on seedlings and blue pollen. To determine the number of ICR events in somatic cells, six-week-old seedlings were stained for GUS activity and the number of blue spots recorded under a binocular microscope. To monitor ICR in pollen, dehiscent anthers of three flowers were combined in 1.5 ml microfuge tube containing 1ml of GUS staining buffer supplemented with 20% methanol to inhibit endogenous GUS activity. The pollen concentration was determined using haemocytometer and the recombination rate was calculated based on the number of blue pollens. Cont…

31. ICR restore a functional GUS gene that can be detected by histochemical staining as blue spots on seedlings (left) and blue pollen (right)

32. results They expressed several bacterial and human recombinases in different combinations in a tobacco transgenic line containing a highly sensitive β-glucuronidase (GUS)-based ICR substrate. Coordinated simultaneous expression of multiple recombinases was achieved using the viral 2A translational recoding system. They found that most recombinases increased ICR dramatically in pollen, where HR will be facilitated by the programmed DSBs that occur during meiosis.

33. DMC1 expression produced the greatest stimulation of ICR in primary transformants, with one plant showing a 1000-fold increase in ICR frequency. Evaluation of ICR in homozygous T2 plant lines revealed increases in ICR of between 2-fold and 380-fold depending on recombinase(s) expressed. By comparison, ICR was only moderately increased in vegetative tissues and constitutive expression of heterologous recombinases also reduced plant fertility. Cont…

34. Conclusions Opportunities for CRISPR/Cas deployment in plant biotechnology are currently limited to gene editing applications but would be greatly expanded by the addition of full gene replacement (gene targeting) technology. Here they show that expression of several bacterial or eukaryotic recombinases or combinations of recombinases can dramatically increase ICR in tobacco. Greatest increases were seen with the single recombinases DMC1, RecG and Rad51. If these stimulations of HR translate to full gene targeting assays where the efficiency of CRISPR/Cas is also deployed to generate targeted double strand breaks, it could pave the way for a revolutionary gene replacement methodology for higher plants.

35. Thank you