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Comparative restriction enzymes analysis of rat chromosomal DNA in vitro and in silico

 

This email address is being protected from spambots. You need JavaScript enabled to view it. , M.A. Abdurashitov, V.N. Tomilov, D.A. Gonchar, S.Kh. Degtyarev

Translated from "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.2, No 3, pp 39-46, 2006

 

Theoretical diagrams of rat chromosomal DNA cleavage at more than 25 different nucleotide sequences have been plotted based on earlier proposed method of restriction enzymes analysis of mammalian genomes in silico. A map of the rat LINE1 repeats digestions at the same nucleotide sequences has been calculated and a correspondence between the diagrams data and LINE1 repeat cleavage positions has been shown. Restriction enzymes analysis in vitro has been performed in order to obtain patterns of rat chromosomal DNA cleavage by endonucleases with respective recognition sequences. A perfect coincidence has been shown between theoretical DNA cleavage diagrams and experimental patterns of DNA hydrolysis with restriction endonucleases.

 

More than 90% of the rat chromosomal DNA primary structure has been already determined [1], and these data are constantly updated. In our previous work, we proposed a simple method of restriction enzymes analysis of mammalian DNA in silico by construction the distribution diagrams of DNA fragments produced by cleavage of chromosomal DNA at recognition sites of restriction endonucleases. Such cleavage diagrams of rat, mouse and human chromosomal DNAs have been plotted for DNA sequences 5'-CCWGG-3', 5'-GATC-3', 5'-GGCC-3' and 5'-CCGG-3'. The comparison of theoretical calculations with experimental results on chromosomal DNA hydrolysis with restriction endonucleases Bst2UI, Kzo9I, HaeIII and MspI which have these recognition sites demonstrated a good correspondence between restriction patterns in vitro and in silico [2]. In the present paper we study rat chromosomal DNA cleavage at a wider range of restriction endonucleases recognition sites.
The goal of the present work is to construct the fragments distribution diagrams produced by cleavage of rat chromosomal DNA at more than 25 recognition sites, including 4-, 5- and 6-nucleotide sequences, and to compare the calculation data with the results of DNA hydrolysis with respective restriction endonucleases.

 

MATERIALS AND METHODS

 

Rat chromosomal DNA

Male Sprague-Dowley rats aged 3-4 months (Breeding Laboratory of Experimental Animals, Institute of Cytology and Genetics, Novosibirsk) were used in the experiments. Genomic DNA from the animal liver was isolated according to [3] with some modifications described below.

Rats were decapitated, liver dissected, and tissue immediately frozen in liquid nitrogen. Pieces of tissue were grinded to a powder under liquid nitrogen using a porcelain mortar and pestle. 0.5 - 1 g of powder were placed into 50 ml cone flask with 5 ml of lysis buffer (100 mM Tris HCl pH 8.0, 10 mM EDTA; 0,5% SDS; 40 μg/ml Proteinase K). The mixture was agitated gently and incubated at 37°C for 3 hrs. 5 ml of phenol (saturated with 10mM Tris HCl pH 8,0) were added. The mixture was incubated for 15 min at room temperature with constant agitation and then centrifuged at 4000 rpm for 5 min at 20°C. The upper aqueous phase was transferred into a cone flask with a pipette using a scissored tip, extracted with a mix of 2,5 ml phenol (saturated with 10mM Tris HCl, pH 8,0) and 2,5 ml chloroform/isoamyl alcohol (24:1) at room temperature for 15 min with constant agitation and centrifuged at 4000 rpm for 5 min at 20°C. The aqueous phase was collected and extracted with 5 ml chloroform/isoamyl alcohol (24:1) as described above with subsequent centrifugation. Then aqueous phase was placed into 50 ml glass and cooled on ice. At slow stirring, isopropanol cooled to 0°C was added drop-wise and DNA precipitated spooled on glass rod, washed in 70% ethanol, gently dried with sterile air (3-6 min) and had been dissolving in 1-1,5 ml of sterile TE buffer (10mM Tris HCl pH 7,0; 1mM EDTA) 24 hours at 4°C.

 

Chromosomal DNA hydrolysis

The following restriction endonucleases manufactured by SibEnzyme Ltd. were used in the work (the recognition site of respective restriction enzymes is given in the brackets):
1) PvuII (5'-CAGCTG-3'), 2) VspI (5'-ATTAAT-3'), 3) PciI (5'-ACATGT-3'), 4) Ksp22I (5'-TGATCA-3'), 5) HindIII (5'-AAGCTT-3'), 6) EcoRI (5'-GAATTC-3'), 7) Bpu10I (5'-CCTNAGC-3'and 5'-GCTNAGG-3'), 8) PctI (5'-GAATGC-3' and 5'-GCATTC-3'), 9) Bse3DI (5'-GCAATG-3'and 5'-CATTGC-3'), 10) SfaNI (5'-GCATC-3' and 5'-GATGC-3'), 11) Bse21I (5'-CCTNAGG-3'), 12) BstV2I (5'-GAAGAC-3' and 5'-GTCTTC-3'), 13) Bst6I (5'-CTCTTC-3'and 5'-GAAGAG-3'), 14) MspI (5'-CCGG-3'), 15) AluI (5'- AGCT-3'), 16) Kzo9I 5'-GATC-3'), 17) TaqI (5'-TCGA-3'), 18) RsaI (5'-GTAC-3'), 19) FatI (5'-CATG-3'), 20) Tru9I (5'-TTAA-3'), 21) Sse9I (5'-AATT-3'), 22) BstDEI (5'-CTNAG-3'), 23) HaeIII (5'-GGCC-3'), 24) AspS9I (5'-GGNCC-3'), 25) Fsp4HI (5'-GCNGC-3'), 26) BspACI (5'-CCGC-3' and 5'-GCGG-3'), 27) HpaII (5'-CCGG-3'), 28) HspAI (5'-GCGC-3'), 29) BstFNI (5'-CGCG-3')
Before hydrolysis reaction, all DNA preparations were treated with ribonuclease A (0.1 mg/ml) for 10 minutes at a room temperature and dialyzed in DispoDialyzer MWCO 50,000 tubes ("Sigma", USA) TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) 100 times of the volume of single DNA preparation at 4°C for 20 hours.
Hydrolysis reactions were performed in 40 μl of the reaction mixture containing 6 μg of DNA, SE-buffers recommended by the manufacturer and 3 μl of restriction enzyme at optimal temperatures for 3 h.

 

Electrophoresis

Electrophoresis in 8% polyacrylamide gel was used to separate DNA fragments from 40 to 500 bp (6 μg of hydrolyzed DNA was applied on gel in each run); 1.5% of low melting point agarose ("Sigma", USA) was used to separate DNA fragments in the range of 200-2000 bp. Electrophoresis in 1% agarose "Type I-A, Low EEO" ("Sigma", USA) was used to separate DNA fragments of higher molecular weight. 3 μg of hydrolyzed DNA was applied on agarose gel in each run. Tris-acetate buffer was used for electrophoresis in all the cases. After electrophoresis DNA bands were stained with ethidium bromide and photographed in UV light.



Data on the nucleotide sequence

The rat DNA sequence was obtained from the resource ftp://ftp.ensembl.org/pub/ (version of June 2, 2006).



Software

The DNA fragments distribution diagrams were represented in the form of the dependence of the total mass of fragments with fixed length (in base pairs) on the fragments lengths (in base pairs) according to the previously described technique [1].

 

RESULTS AND DISCUSSION

Previously we have presented fragments distribution diagrams of rat chromosomal DNA cleavage at recognition sites of restriction enzymes HaeIII (5'-GGCC-3'), MspI (5'-CCGG-3'), Kzo9I (5'-GATC-3') and Bst2UI (5'-CCWGG-3'). Good correlation between the calculation data and the results of experiments on DNA hydrolysis with these restriction enzymes has also been demonstrated [2]. In this work we plotted the fragments distribution diagrams of rat chromosomal DNA digestion at a wider range of recognition sites including six nucleotides sequences. In accordance with the earlier proposed method [2], the distribution diagrams were constructed in the arctangensoid scale, which imitates the separation of the DNA fragments by electrophoresis in agarose gel. Fig.1a shows the diagrams of DNA cleavage mainly at six nucleotides sequences, which are recognition sites of both well-known restriction enzymes (HindIII and EcoRI) and ones rarely used (SfaNI and some others). These diagrams were selected based on the presence of peaks of 5.5 and more million base pairs, a threshold value for appearance of respective bands in the analysis of the products of DNA cleavage by electrophoresis in agarose gel [2]. Table 1 summarizes data on 26 such peak fragments. As Table 1 shows, in most cases the presence of peaks of such height is explained by the presence of several fragments with sizes differing by 1-2 nucleotides for which, in accordance with the proposed method [1], the total peak height value was calculated. The formation of clusters of such fragments with minimal differences in the sizes is probably associated with deletions and insertions in DNA repeats [4]. Cleavage of these DNA repeats results in formation of the bands observed on agarose and acrylamide gels [2], [5, 6, 7]. Direct analysis of DNA repeats seems to be a simpler method of obtaining a digestion pattern in silico, however, its use may be incorrect due to rather high variability of the nucleotide sequence in these DNA regions. For comparison, we carried out analysis of the averaged primary structure of the most complete (6914 bp) LINE1 repeat of rat DNA [8]. Data on the length of restriction fragments of this consensus LINE1 repeat, which corresponds to the data of the column 3, are presented in the fourth column of Table 1. As the Table shows, a sufficiently high correlation of genomic DNA and consensus LINE1 repeat cleavage is observed. However, for a number of sites, the calculation data obtained with our method differ from those of LINE1 repeat analysis. In particular, one of VspI sites was omitted in the latter case; a difference is observed in the sizes of PvuII fragment and upper fragments of Ksp22I and SfaNI patterns of DNA digestion. This difference is associated with extra 3 bp and 133 bp length fragments, which are inserted in consensus LINE1 repeat fragment. A comparative study of consensus LINE1 repeat nucleotide sequence and some other ones [9] shows that these short DNA fragments indeed present in some LINE1 repeats, but an analysis of the whole DNA by our method as well as experimental data don't reveal these fragments. A revised version of consensus LINE1 repeat nucleotide sequence [10] includes 1) deletion of 133 bp fragment at positions 1294-1426, 2) deletion of 3 bp fragment at positions 1278-1280 and 3) replacement of A for T in position 1438 with formation of the second VspI site. Data on cleavage of new consensus LINE1 repeat proposed in this work [10] are given in 5th column. Comparison of 3rd and 5th columns shows a perfect coincidence of data provided and confirms a correct DNA sequence of proposed LINE1 repeat if compare to published one.
In general, however, the analysis of consensus LINE1 repeat does not allow to evaluate the number of obtained DNA fragments of the given length and thus to predict their visualization in the experiment. For example, low-molecular weight fragments in EcoRI, SfaNI, BstV2I and some other restriction enzymes digestions deduced from the analysis of LINE1 repeat structure give low peaks in the diagrams and, as it will be shown further, can't be seen in the gel photographs.

 

Restriction endonuclease Recognition site Lengths of the peak fragments* Lengths of fragments* produced by cleavage of L1_RN [17] Lengths of fragments* produced by cleavage of consensus L1 repeat (this work)
PvuII CAGCTG 4549,4550 4685 4549
VspI ATTAAT 3361,3362 - 3362
PciI ACATGT 1171,1172 1172 1172
Ksp 22 I TGATCA 2678,2679
2094,2095
2814
2095
2678
2095
HindIII AAGCTT 3626,3627
179
3627
179
3627
179
EcoRI GAATTC 2316,2317,2318
1372,1373
2318
1373
2318
1373
Bpu 10 I CCTNAGC 2116,2117 2117 2117
PctI GAATGC 2105,2106
641, 619
413
236
2106
641 , 625 **
413
236
2106
641, 625**
413
236
Bse3DI GCAATG 3908,3909 3917 ** 3917**
SfaNI GCATC 2786,2787
1151
437
2922
1170 **
418 **
2786
1170**
418**
Bse21I CCTNAGG 2140,2142
1960,1961,1962
2142
1962
2142
1962
BstV2I GAAGAC 1279,1280
924
747
327
1266 **
924
761 **
327
1266**
924
761**
327
Bst 6 I CTCTTC 2656,2657
2115,2116
2657
2116
2657
2116

Table 1. Predicted lengths of DNA fragments, which are produced by cleavage of rat genome, published and proposed consensus sequences of LINE1 repeat.
* - included only fragments less than 500 bp with peak values more than 4 millions bp and fragments more than 500 bp with peak values more than 5.5 millions bp
The lengths of fragments in fourth and fifth columns, which are the same as the length in third column, are shown in bold.
** - the length difference of indicated DNA fragments, which are produced by cleavage of IIS type restriction endonucleases PctI, Bse3DI, SfaNI and BstV2I, is caused by a location of the restriction enzymes cleavage positions outside recognition sequences


Fig.1b presents experimental data on rat chromosomal DNA cleavage with restriction endonucleases. The comparison of calculation data in the third column of Table 1 with experimental results shows that all the indicated fragments are present in the form of bands in agarose gel photographs.

 

b_320_200_16777215_00_Pics_paper28_fig1.jpg

Fig. 1.

a) Distribution diagrams of total DNA fragments lengths (in bp) depending on the fragment size for restriction endonucleases recognition sites. M1 - calculated DNA fragment length marker corresponding to SE 1 kb ladder. Detailed diagrams are shown on the Fig 4.
b) b) Electrophoregram of rat genomic DNA digestion with restriction endonucleases in 1% agarose gel. M1 - DNA fragment length marker SE 1 kb ladder.
c) c) Electrophoregram of rat genomic DNA digestion with restriction endonucleases in polyacrylamide gel. M2 - DNA fragment length marker pUC19/MspI.


In particular, the presence of bands of respective molecular weight at rat DNA hydrolysis with restriction enzymes PvuII, VspI, PciI, Ksp22I, HindIII, EcoRI, Bpu10I, PctI, Bse3DI, SfaNI, Bse21I, BstV2I and Bst6I is experimentally observed. The patterns of chromosomal DNA hydrolysis with restriction enzymes BamHI and PceI, respectively, are presented in Fig.1b on runs 4 and 15. The names of these enzymes are absent in Table 1, as cleavage at recognition sites BamHI and PceI does not provide diagrams with high peak values. However, as it is seen from Figure 1b, DNA hydrolysis with these restriction enzymes also results in the appearance of DNA band of 4500-5000-bp length, which is especially noticeable in the case of PceI. Besides, DNA hydrolysis with Bst6I provides an additional band of 4500-5000 base pairs length presented as a small peak in Fig.1a. Detailed analysis of DNA cleavage diagrams at recognition sites of restriction enzymes BamHI, Bst6I and PceI shows that in all three cases several fragments, which differ in size in the range of 200 bp and have low peak values (data are not presented), are located in the region of 4500-5000 base pairs. The appearance of the band on the gel is probably associated with low resolution of electrophoresis for fragments of 4500-5000 base pairs length and can result from several small peaks overlapping. Another consequence of such overlap in peaks is the thickness of the upper band of DNA hydrolysis with Ksp22I which corresponds to 2678-2679-bp fragments length.
From Fig.1a and Table 1 it can be seen that in accordance with the obtained diagrams, the products of rat DNA cleavage with restriction enzymes HindIII, PctI, SfaNI and BstV2I include low-molecular weight fragments. To analyze them, electrophoresis in PAAG was performed. This electrophoresis visualizes fragments with a smaller peak value because a larger amount of DNA is loaded on the gel run. Fig.1c shows the patterns of DNA hydrolysis with restriction endonucleases HindIII, PctI, SfaNI and BstV2I in PAAG. DNA cleavage with restriction enzyme HindIII results in the formation of the 179-bp fragment, which is present in Table 1, and an additional fragment of 370 base pairs. The last result is attributed to a hydrolysis of satellite DNA, which contains a recognition site of restriction enzyme HindIII [6]. In Fig.1c, one can also see all the other low molecular weight fragments, which are present in the third column of Table 1. PctI forms a double of 641 and 619 as well as fragments of 413 and 236 base pairs, and SfaNI cleaves off the 437-bp fragment and BstV2I - the 327-bp fragment. The other low molecular weight fragments, revealed in the analysis of LINE1 repeat for recognition sites of restriction enzymes are not detected in the experiment. This fact does not count in favor of using the repeats data in construction of DNA cleavage diagrams.

 

 

b_320_200_16777215_00_Pics_paper28_fig2.jpg

Fig. 2.

a) Distribution diagrams of total DNA fragments lengths (in bp) depending on the fragment size for restriction endonucleases recognition sites. M1 - calculated DNA fragment length marker corresponding to SE 1 kb ladder. Detailed diagrams are shown on the Fig 5.
b) b) Electrophoregram of rat genomic DNA digestion with restriction endonucleases in 1% agarose gel. M1 - DNA fragment length marker SE 1 kb ladder.
c) c) Electrophoregrams of rat genomic DNA digestion with restriction endonucleases RsaI and AspS9I in polyacrylamide gel. M2 - DNA fragment length marker pUC19/MspI.


Fig.2a shows diagrams of DNA cleavage mainly at four nucleotides sequences; the numbers shown on them indicate the sizes of fragments with peak values of more than 5.5 million bp. As compared with diagrams in Fig.1, the formation of a considerably larger number of DNA fragments is observed in this case, which results in the appearance of the basic curve of the fragments distribution and peaks against this background. Data on these fragments peaks are collected and compared with corresponding results of simulation of LINE1 consensus fragment cleavage in Table 2.

 

Restriction endonuclease Recognition site Lengths of peak fragments Lengths of fragments* formed by L1_RN [17] cleavage Lengths of fragments* formed by cleavage of consensus L1 repeat sequence proposed in this work
MspI CCGG 5537, 5538
404, 405
5673
405
5537
404
AluI AGCT 1127 1127 1127
Kzo9I GATC 768
752
449, 447
337
321
768
752
449
337
321
768
752
449
337
321
TaqI TCGA 1233
1187
1233
1187
1233
1187
RsaI GTAC 1650
802
555
330
197
1650
802
555
330
197
1650
802
555
330
197
FatI CATG 1007
832
648
634
1007
832
648
634
1007
832
648
634
Tru9I TTAA 703
573, 574, 575
468
447
703
574
468
447
703
573
468
447
BstDEI CTNAG  
624
624  
624
HaeIII GGCC 1173
 
863
697
588
575
1173
863
697
588
575
1173
 
863
697
588
575
AspS9I GGNCC 1110
1025
 
554
534
456
374
353
202
155
1110
1025
554
534
456
374
353
202
155
1110
1025
 
554
534
456
374
353
202
155
Fsp4HI GCNGC 1613
 
1035
825
543
1613
1035
825
543
1613
 
1035
825
543
Bst2UI CCWGG 1690, 1691
1017
713
462
341
313
291
215
177
146
1691
1017
713
465
341
313
291
215
177
146
1691
1017
713
461
341
313
291
215
177
146

 

Table 2. Comparison of predicted lengths of fragments formed due to cleavage of rat genome with several restriction enzymes with calculated lengths of fragments formed by cleavage of consensus sequences od LINE1-repeats.
* - only fragments <500 b.p. in length with peak values more than 4 billions b.p. and fragments >500 b.p. in length with peak values more than 5.5 billions b.p. are included.
Fragments in the fourth and fith columns which are of the same length as those in the third column are shown in bold.


The Table 2 data show that the peaks formation is associated with the cleavage of LINE1 repeats, whereas the presence of the basic curve is the result of cleavage of the rest of genomic DNA. The form of this curve varies depending on the analyzed nucleotide sequence, e.g., for recognition sites of restriction enzymes AluI, FatI, Tru9I , Sse9I and BstDEI the number of fragments in the length range from 50 to 500 bp is considerably higher than for the others. Fig.2b presents the pattern of rat DNA hydrolysis with restriction endonucleases possessing recognition sites presented in Fig. 2a. The comparison of Figures 2a and 2b shows that the presence of the basic curve of the fragments distribution in the diagrams correlates with the location of DNA spot in the corresponding photographs of gels. The formation of such spot complicates the visualization of individual DNA fragments, which have peak values in the diagrams. Because of this, the numbers on the diagrams in Fig.2a indicate the sizes of only those fragments, which are outside these dark areas. A comparison of data presented in Figures 2a and 2b reveals a correspondence between more than 25 peak values of the fragments lengths obtained in the diagrams and the bands seen in the gel photographs. In particular, as was shown previously [2], hydrolysis with restriction enzymes MspI and HaeIII reveals clearly distinguishable bands, which correspond to peaks of 5537+5538 and 404+405 bp in the first case, and 1173, 863, 697, 575+588 as well as 370 bp (satellite DNA) in the second case. The appearance of bands corresponding to the 1127-bp fragment, the double fragment (752 and 768 bp) and the double peak (1187 and 1233 bp) is clearly observed when DNA was treated with restriction enzymes AluI, Kzo9I and TaqI, respectively. DNA hydrolysis with enzymes RsaI, FatI and Tru9I provides several weak bands corresponding to the peaks at 1650, 989, 802 and 555 in the first case, 1007 and 832 in the second case and 703, 573-575 and 447-468 bp in the third case. DNA hydrolysis with restriction enzymes Sse9I and BstDEI does not provide clearly defined bands due to their overlapping with the basic curve of low molecular weight fragments. Cleavage with restriction enzyme AspS9I results in the appearance of discrete bands corresponding to the peaks at 1025+1100 bp and 534+554 bp, while restriction enzyme Fsp4HI provides the 1613 bp fragment and less noticeable fragments (1463, 1035, 825 and 543 bp). As seen in Fig.2a, low values of the basic curve in the region of less than 600 bp, which are required for visualization of fragments in PAAG, are observed in the case of DNA cleavage at recognition sites of restriction enzymes AspS9I, Fsp4HI, HaeIII, Kzo9I, MspI, RsaI and TaqI. The results of electrophoresis of the products of rat chromosomal DNA cleavage with restriction enzymes HaeIII, Kzo9I and MspI in PAAG were discussed previously [2]. As for the other restriction enzymes the corresponding peaks exist in diagrams of DNA cleavage with enzymes AspS9I and RsaI only. Fig.2c presents data of electrophoresis of the products of rat DNA cleavage with restriction enzymes AspS9I and RsaI in PAAG. The first case reveals bands corresponding to the 456, 374, 353, 202 and 155 bp fragments and the second case shows bands which correspond to 581, 555 bp fragments plus an additional 370 bp fragment of satellite DNA [6].
From data presented in Fig.2b, it can be seen that DNA cleavage with restriction endonucleases TaqI, RsaI and FatI , which all have recognition sites with the same GC composition, results in different depth of hydrolysis and different location of the DNA spot. Correspondingly, basic curves for recognition sites of these restriction enzymes, as shown in Fig.2a, also considerably differ from each other. One can point out several peculiarities of rat chromosomal DNA, that influence the depth of hydrolysis by restriction endonucleases with the same set of nucleotides in the recognition site.

 

Расщепление геномной ДНК крысы эндонуклеазами рестрикции

 

 

Fig. 3.
Rat genomic DNA digestion with restriction endonucleases containing CG-dinucleotide in their recognition sites. M1 - DNA fragment length marker SE 1 kb ladder.


1. Hindrance of genomic DNA hydrolysis due to CG-dinucleotides methylation. According to the available estimates, 70-80% of all CG pairs in mammalian genomes are methylated [11]. Fig.3 shows the results of DNA cleavage with restriction endonucleases containing dinucleotide CG in the recognition site, in particular, with restriction enzymes BspACI : (5'-CCGC-3' and 5'GCGG-3'), BstFNI (5'-CGCG-3'), HspAI (5'-GCGC-3'), HpaII and MspI (5'-CCGG-3'). As seen in Figure 3, enzymes BspACI , BstFNI, HspAI and HpaII only slightly cleave genomic DNA, whereas MspI, which is able to cleave the site 5'-CmCGG-3', hydrolyzes DNA very well. Thus, out of all analyzed restriction enzymes with CG dinucleotide in the recognition site, chromosomal DNA is effectively cleaved by MspI and TaqI, with the latter also being insensitive to cytosine methylation in the recognition site (Fig. 2b and 3).
In addition, DNA hydrolysis with some restriction enzymes can be complicated by recognition sites overlapping with CG-methylated dinucleotide. When compared with the location of the basic curve in corresponding diagrams, the difference in dark area positions on photographs of DNA products separation after cleavage by HaeIII and Fsp4HI may be explained by presence of 5mCG dinucleotide (Fig 2b). Part of recognition sites of restriction enzymes HinfI (5'-GANTC-3') and EcoRI (5'-GAATTC-3') in rat satellite DNA contains methylated cytosine and these sites are hydrolyzed at a considerably lower rate than the non-modified recognition sites [5].

2. GC composition. GC composition of rat genomic DNA is 40% [12] and restriction enzymes with AT recognition sites (Tru9I and Sse9I) should produce a deeper hydrolysis than restriction endonucleases which have GC pairs only in the recognition site (for example, HaeIII and AspS9I). Data provided in Fig.2 confirm this consideration.

3. The presence of high-frequency and low-frequency dinucleotides in the recognition site. The occurrence frequency of some dinucleotide pairs considerably differs from the average statistical expected values. This can account for the difference in the average length of the restriction fragments at DNA hydrolysis with enzymes whose recognition sites have the same GC composition.
Thus, DNA hydrolysis with restriction endonuclease TaqI (recognition site TCGA) produce the area of chromosomal DNA fragments spot is considerably higher than that at hydrolysis with restriction enzymes Kzo9I (GATC) and RsaI (GTAC) though GC composition of recognition sites of all these enzymes is the same.
This result can be accounted for by the fact that in chromosomal DNA of eukaryotes the occurrence frequency of dinucleotide CG is five times lower than the average statistical expected frequency [13]. Low occurrence frequency of the CG pair can be explained by a high rate of transformation of 5-methylcytosine in this dinucleotide into thymine which occurs as a result of the spontaneous deamination reaction [14].
Similarly, one can explain the difference in the depth of DNA hydrolysis with restriction enzymes HaeIII (recognition site 5'-GGCC-3') and MspI (recognition site 5'-CCGG-3'), as shown in Fig.1b, although recognition sequences of both enzymes consist of only GC pairs. MspI recognition site contains low-frequency dinucleotide CG and enzyme's action results in a smaller depth of DNA hydrolysis if compared to that of HaeIII hydrolysis.
A contrary situation is observed for dinucleotides TG, CA, AG and CT. These dinucleotides in chromosomal DNA of eukaryotes are represented with higher frequency compared to the average statistical value of occurrence [15, 16]. This can explain the deep hydrolysis, observed in Fig.2b for restriction enzymes FatI, (recognition site CATG), AluI(recognition site AGCT) and BstDEI (recognition site CTNAG). The recognition site of these restriction endonucleases contains two high-frequency dinucleotides (CA and TG for FatI, AG and CT for AluIand BstDEI) at a time.

 

b_160_120_16777215_00_Pics_paper28_rat_PvuII.gif b_160_120_16777215_00_Pics_paper28_rat_VspI.gif b_160_120_16777215_00_Pics_paper28_rat_PciI.gif b_160_120_16777215_00_Pics_paper28_rat_Ksp22I.gif
PvuII VspI PciI Kzo22I
b_160_120_16777215_00_Pics_paper28_rat_HindIII.gif b_160_120_16777215_00_Pics_paper28_rat_EcoRI.gif b_160_120_16777215_00_Pics_paper28_rat_Bpu10I.gif b_160_120_16777215_00_Pics_paper28_rat_PctI.gif
HindIII EcoRI Bpu10I PctI
b_160_120_16777215_00_Pics_paper28_rat_Bse3DI.gif b_160_120_16777215_00_Pics_paper28_rat_SfaNI.gif b_160_120_16777215_00_Pics_paper28_rat_Bse21I.gif b_160_120_16777215_00_Pics_paper28_rat_BstV2I.gif
Bse3DII SfaNI Bse21I BstV2I
b_160_120_16777215_00_Pics_paper28_rat_Bst6I.gif b_160_120_16777215_00_Pics_paper28_rat_BamHI.gif b_160_120_16777215_00_Pics_paper28_rat_PceI.gif  
BstV6II BamHI PceI  

Fig. 4. Detailed distribution diagrams of total DNA fragments lengths depending on the fragment size for restriction endonucleases recognition sites.

 

b_160_120_16777215_00_Pics_paper28_rat_MspI.gif b_160_120_16777215_00_Pics_paper28_rat_AluI.gif b_160_120_16777215_00_Pics_paper28_rat_Kzo9I.gif b_160_120_16777215_00_Pics_paper28_rat_TaqI.gif
MspI AluI Kzo9I TaqI
b_160_120_16777215_00_Pics_paper28_rat_RsaI.gif b_160_120_16777215_00_Pics_paper28_rat_FatI.gif b_160_120_16777215_00_Pics_paper28_rat_Tru9I.gif b_160_120_16777215_00_Pics_paper28_rat_Sse9I.gif
RsaI FatI Tru9I Sse9I
b_160_120_16777215_00_Pics_paper28_rat_BstDEI.gif b_160_120_16777215_00_Pics_paper28_rat_HaeIII.gif b_160_120_16777215_00_Pics_paper28_rat_AspS9I.gif b_160_120_16777215_00_Pics_paper28_rat_Fsp4HI.gif
BstDEI HaeIII AspS9I Fsp4HI

Fig. 5. Detailed distribution diagrams of total DNA fragments lengths depending on the fragment size for restriction endonucleases recognition sites.

 

CONCLUSION


Diagrams of rat chromosomal DNA cleavage at 25 recognition sequences of restriction endonucleases have been plotted and discussed. The presence of more than fifty significant peaks of DNA fragments (or clusters of fragments) has been revealed for a cleavage at these nucleotide sequences. A map of LINE1 consensus repeat digestion with restriction enzymes has been produced and a high correspondence between the experimental data displayed in diagrams and LINE1 repeat's cleavage positions has been shown. Based on restriction enzymes analysis a revised version of consensus LINE1 repeat sequence has been suggested. Experiments on chromosomal DNA hydrolysis with restriction endonucleases and subsequent electrophoresis of reaction products in agarose and/or polyacrilamide gels have been performed. A comparison of the constructed distribution diagrams of rat chromosomal DNA fragments and the patterns of DNA digestions with the corresponding restriction endonucleases has revealed a good correspondence between theoretical and experimental data.

The authors thank Dr. V. I. Kaledin Dr. and Dr. G. V. Vasilyev for assistance in animal experiments and DNA isolation.

 

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ACCEPTED ABBREVIATIONS

RE - restriction endonuclease
bp - base pair
EDTA - ethylene diamine tetraacetic acid
PAAG - polyacrylamide gel