The positively charged central groove that run across the dimmer interphase possesses the correct dimensions to function as the binding site tyrosines are juxtaposed with the promoters at the dimmer interface would be expects to create a DNA gate for the transport reaction.
The homologous yeast spo11 protein is involved in producing double strand breaks that initiate the process of meiotic recombination, but unlike a topoisomerase,the enzyme dose not rejoin the two ends and is instead cleaved away from the DNA thus, the spo11protein functionally behave like a type II topoisomerase that is unable to close the gate after cleavage. One possible explanation for the properties of spo11is that spo11 lacks a functional analogue of the topoisomerase VIB subunit bridge and cannot prevent dissociation of the A subunit. If the above hypothesis is correct, then is seems likely that association with one regulates the cleavage activity of spo11 or more accessory proteins, none of which provide abridge between the two A subunits. Any combination of the nine other proteins required in yeast for producing double-strand breaks during meiosis could function in this regard.
The molecular techniques such as plasmid isolation, restriction digestion, ligation, bacterial transformation etc. were based on protocols described by Sam brook et.al. (2001). Stock solutions of various reagents were prepared in RO water. Stocks of various antibiotics were prepared in DMSO and stored at -20 c. Water needed for PCR reactions, restriction digestion, precipitation of DNA etc were obtained from Elix(tm), Millipore water purification systems, France. All aseptic procedures were carried out under sterilized conditions in laminar flow hoods. (Kartos international, India)
CHEMICAL REAGENTS AND GLASSWARES USED
General use chemicals were of AR grade procured from Qualigens, Difco Hi media (India). Antibiotics and other fine chemicals were purchased either from Sigma Chemical Co. St. Louis, USA. Nitrocellulose paper (Hydrogen C), and Mega prime labeling kit were obtained from Amersham, USA. Restriction enzymes were obtained either from Roche Molecular Biochemicals, Germany or MBI Fermentas. USA. All the glass wares used was purchased from Borosil, India. Plastic wares such as micro tips, micro centrifuge tubes, Petri plates etc. were obtained from Tarson, India or Axygen USA.
RICE GENOMIC DNA ISOLATION
Total DNA was extracted from plant tissue following the procedure of Dellaporta et.al. (1983) with slight modifications. Around 3 gm of shoot portion was taken from 4 days old etiolated ice seedlings, so as to minimize chloroplast and mitochondrial DNA contamination. The tissue was ground to fine powder in liquid nitrogen using precooled mortar and pestle with the help of liquid nitrogen .The powdered tissue was transferred to precooled SS-34 tube and 15 ml of extraction buffer (100Mm Tris -Cl, pH 8.0, 50.0mM EDTA,PH 8 ,500Mm NaCl and 10mM B-ME)added immediately . The tissue was homogenized by vortexing followed by the addition of 1 ml of 20% SDS.
The tubes were kept at 65 for 20min. with intermittent vigorous shaking. Subsequently, 5.0ml of 5M potassium acetate was added and the tube incubated on ice for 30min. After vigorous shaking. The tube was then centrifuged at 15,000 rpm for 20min at 4ï¿½c in a centrifuge. The supernatant was carefully transferred to a fresh SS-34 tube through two layers of autoclaved muslin cloth and 10ml isopropanol added, properly mixed and incubation carried out at -70 C for 1 hr. The DNA was pelleted by centrifugation at 12,000 rpm for 15 min at 4C in Sorvall SS-34 rotor. The pellet was suspended in 0.7 ml of high salt buffer (50 mM Tris-Cl, pH 8.0 and 10 mM EDTA, pH 8.0) and kept for dissolving in ice for 10-15 min. This step onwards wide bore tips (1 ml cut tips) were used for pipeting the genomic DNA in order to avoid shearing. The liquid was transferred to a microfuge tube and centrifuged at 13,000 rpm for 10 min at 4 C.
The supernatant was transferred to a fresh microfuge tube and undissolved debris discarded. 6ï¿½l of RNase (10ï¿½g/ï¿½l) was added and incubated at 37 C for 1 hour. Subsequently, 350of phenol and 350 of chloroform was added, mixed properly and then centrifuged at 10,000rpm, 4ï¿½C for 10 minutes. The aqueous phase was taken in fresh MCT and 700 of chloroform was added, mixed gently and centrifuged at 10,000rpm, 4 C for 10 minutes. Aqueous phase was taken and 1/10th volume of 3M sodium acetate, pH 5.2, and 0.7 volume of isopropanol were added and mixed gently to precipitate the DNA and kept at room temperatures. The DNA was pelleted at 10,000rpm for min, washed with 70% ethanol and dried in speed Vac TM (Savant Instruments Inc., NY). The pellet was dissolved in minimum volume of 1X TE buffer, PH 8.0.
The DNA was quantified spectrophotometrically (DU640B spectrophotometer, Beckman Instruments, USA) in a quartz cuvette by measuring absorbance of 1ï¿½l aliquot, diluted with 1ml with RO water, take O.D at 260nm to quantitate the DNA. And also take O.D at 280nm to qualitate the DNA.check the DAN quality by loading on 0.8% agarose gel. Absence of smear below the band was taken as the criteria for selecting high molecular weight DNA. The polymerase chain reaction can also be used to obtain a pure sample of a gene. This is because the region of the starting DNA molecule that is copied during PCR is the segment whose boundaries are marked by the annealing positions of the two oligonucleotide primers if the primers anneal either side of the gene of interest, many copies of that gene will be synthesized.