Control del tiempo de refrigerio en los operadores

II.15.A. Nick translation of DNA fragments

Purified DNA restriction fragments, isolated as described in section II.8.B., were labelled by nick translation using a modification of the method of Rigby et al (1977). Initially, a 30 ul reaction mixture was assembled containing the following components:- 100 ng of purified DNA, 3 ul of 10 x nick translation buffer (0.5 M tris-HCl pH 7.8, 50 siM HgCl2 t 100 mM dithiothreitol), 1 ul of 100 uM dATP, 1 ul of 100 uM dTTP, 1 ul of 100 uM dCTP, 1 ul of 1 uH dGTP and 1-6 ul of (-C-32P)dGTP (>3 000 Cl/mol), (th. amount depending on the required specific activity of the probe). To this mixture was added 1 ul of freshly prepared 50 ug/ml DNAse I (from bovine pancreas). Following incubation for 15 min at room temperature, 1 ul of E. coli DNA polymerase I (Kornberg) was added. The preparation was then gently mixed and incubated for 3 h at 15°C. Removal of the unincorporated nucleotides was achieved by spermine precipitation. To this end, 3 ul of 100 mM spermine-HCl and 5 ul of 1 ug/ul E. coli tRNA were added to the preparation which was then lightly vortexed. The tube was placed on ice for 15 min and then centrifuged for 10 min in a microcentrifuge to pellet the nucleic acids. The pellet was washed twice with 100 ul of 70Z (v/v) ethanol, 0.3 M sodium acetate pH 8.5, 1.25 mM EDTA,

twice with 100 ul of 70X (v/v) aqueous ethanol, dried in vacuo and redissolved in 100 ul of sterile distilled water. This nick translation method was used to prepare DNA probes for the screening of the bacteriophage lambda genomic libraries and most of the Southern biota presented.

II.15.B. Oligolabelling of DNA fragments

A modification of the oligolabelling method of Feinberg and Vogelstein (1984) was used to prepare ^2p-

radiolabelled DNA probes of high specific activity. Such probes were used for hybridisation to both Northern blots and some genomic Southern blots. Initially a preparation of the DNA fragment of interest, purified as described in section II.8.B., was denatured by incubation for 5 min in a boiling water bath. The preparation was then

transferred to a water bath at 37°C for 5-10 min. Meanwhile the remainder of the reaction mixture was assembled in a 1.5 ml Eppendorf tube. The mixture consisted of 3 ul of 5 x oligolabelling buffer (0.24 M tris-HCl pH 8.0, 25 mM M g C ^ , 50 mM 2-mercaptoethanol, 0.1 mM dATP, 0.1 mM dTTP, 0.1 mM dCTP, 1 M HEPES pH 6.6, 54 ^260 units/ml hexadeoxyribonucleotides (from calf thymus DNA), 0.6 ul of 10 mg/ml nuclease-free bovine serum albumin, 2-5 ul of (*-32P)dGTP (>3 000 Cl/mmol) and 0.6 ul of 3.5 u/ul DNA polymerase I Klenow fragment. A 10 ng

aliquot of the DNA preparation was added to this mixture and the final volume was adjusted to 15 ul by the addition of sterile distilled water. The preparation was incubated at room temperature for a minimum of 4 h and the

unincorporated nucleotides removed by spermine precipitation as described in section II.16.A.

II.15.C. Radiolabelling of DNA restriction fragments using an end filling reaction

Hind Ill-digested bacteriophage lambda DNA fragments and Hpa II-digested pBR322 plasmid DNA fragments were r.dlolabelled with (K-32P)dGTP and (*<-32P)dCTP

respectively using a modification of the method of Drouin (1980). The bacteriophage or plasmid DNA was first digested with the appropriate restriction endonuclease at a concentration of 0.05 ug/ul. To 20 ul of the digested DNA preparation was added 1 ul of 3.5 u/ul DNA polymerase I Klenow fragment and either 2 ul of (•C-^^P)dGTP (>3 000 Ci/mmol) in the case of Hind Ill-digested bacteriophage

lambda DNA, or 2 ul of («<-32P) dCTP (>3000 Cl/mmol) in the case of Hpa II-digested pBR322 plasmid DNA. The

preparation was then incubated at room temperature for 20 min and extracted once with an equal volume of TE- equilibrated 1:1 (v/v) phenol/chloroform mixture. The DNA was precipitated by the addition of 2.5 ul of 7 M ammonium acetate and 50 ul of ethanol. The precipitate was then

collected by centrifugation for 10 min in a

microcentrifuge, dried in vacuo and redissolved in 300 ul of TE.

SECTION III.l. GENOMIC ORGANISATION OF CASTOR BEAN LECTIN GENES

111.1. A. Estimation of R. communis genome size

It is helpful to know the genome size when attempting to clone specific genes from an organism, since an estimate may be made as to how many recombinant clones must be screened. Unfortunately, the C-value or haploid genome size of R. communis is not available in the literature. An approximate estimate was therefore made be comparing the DNA yields obtained from known numbers of nuclei. Table 4 shows the respective DNA yields and nuclei counts of a number of preparations, as well as the individual and mean C-values calculated. The results shown are discussed in section IV.l.C.i).

11.1. B. Estimated number of lectin genes in the R. co— unis genome

In order to estimate the size of the lectin gene family of R. communis, a genomic Southern blot was performed as described in Figure 5. The blot was hybridised with a ricin cDNA probe and washed at low stringency, so that both ricin and RCA I-like genes would be identified in the resulting autoradiograph. The result, shown in Figure 5

Sample No of nuclei DNA yield Calculated C-value 1 2.1 x 108 180 ug 0.43 pg 2 5.4 x 108 335 ug 0.31 pg 3 5.7 x Ho 00 215 ug 0.19 pg 4 1.4 x 109 650 ug 0.23 pg Mean C-value - 0.26 pg Confidence limits (95X level) ■ ♦ or - 0.17 pg

Table 4 Estimation of R. communi8 C-value from nuclei counts and DNA yields

In order to estimate the R. communis haploid genome size, the DNA yields obtained from preparations containing known numbers of nuclei were quantified. A C-value was calculated in each case and an overall mean value, plus confidence limits, was determined. Although the R. communis seedlings used were of unknown variety, it was assumed that the cultivar used is diploid, as has been shown for other cultivars (D Griffiths, pers. comm.).

Figure 5 Identification of genomic DNA restriction fragments containing lectin-hybridising sequences

Two 10 ug samples of castor bean nuclear DNA were digested with restriction enzymes and electrophoresed on a 1 x TAE, 0.6X (w/v) agarose gel. The gel was photographed under uv illumination following ethidium bromide staining. A Polaroid negative of the gel is shown in tracks 1 to 3. The size markers in track 1 are of Hind III - digested bacteriophage lambda DNA. Tracks 2 and 3 show the patterns obtained when castor bean nuclear DNA is digested with Eco RI and Hind III respectively.

The gel was Southern blotted onto Hybond-N and the membrane was probed with a nick-translated ricin cDNA fragment (sections II.4.C., II.13.A.). After low stringency washing (section II.4.D.), the membrane was autoradiographed. Tracks 4 and 5 show the size distribution of lectin-hybridising fragments in castor bean DNA digested with Eco RI and Hind III respectively.

indicates that the R. communis lectin gene family consists of appcoximtely 8 members. A detailed discussion is given in section IV.4.A.

In order to estimate how many ricin-like genes are present in the R. communis genome, a second genomic blot was probed with the same ricin cDNA fragment, but washed instead at high stringency, as described in Figure 6• The result, which is also discussed in section IV.4.A., appears to indicate that two members of the lectin gene family are ricin-like.

Figure 6 Identification of genomic DNA restriction fragments containing sequences sharing high homology with a ricin cDNA probe

Two 10 ug samples of castor bean nuclear DNA were digested with restriction enzymes and electrophoresed on a 1 x TAE, 0.6X (w/v) agarose gel alongside Hind III - digested lambda DNA size markers. The gel was Southern blotted onto nitrocellulose and probed with a nick- translated ricin cDNA probe (sections II.4.C . ,II.15.A . ). A high stringency wash was then carried out (section II.4.D.) so as to specifically identify DNA fragments containing ricin-like sequences. The position of the size markers is indicated. Tracks 1 and 2 show the patterns of hybridisation obtained from Hind III and Eco RI-digested castor bean DNA respectively.

SECTION III.2. NUMBER AND DIVERSITY OF CLONED LECTIN