Inverse PCR to identify DNA sequence upstream
of the pea HMG I/Y open reading frame

Druffel, K., Carson , J.A.,                                                                                          Dept. of Plant Path.

Hartney, S. and Hadwiger, L.A.                                         Washington State Univ., Pullman , WA , USA

 In the pursuit of the defense responses in pea plants, or in any induced response in eukaryotic organisms, the sequence of the open reading frame of the induced gene is attainable from a cDNA clone. Once the open reading frame transcription start site of the cDNA clone has been obtained, the promoter region is often of interest and is accessible by inverse PCR. Although inverse PCR is a standard technique, all PCR strategies do not necessarily result in success. The promoter of the pea gene HMG I/Y is of interest because of the possibility that its coded protein is influencing its own transcription. The HMG I/Y protein contains four AT-hooks (2, 5) that have affinity for AT-rich regions of DNA (4). The extensive research relative to this property has led to the recognition that HMG-I/Y is an architectural transcription factor (7). It has recently been determined that both its RNA transcript and its protein product are depleted as the pea defense response is initiated (4).

The pea HMG I/Y protein can efficiently bind to AT-rich segments of promoters from pea PR genes, genes that are activated as the pea tissue resists fungal pathogens (1, 4).  Therefore it was of interest to determine if such AT-rich regions were also present in the HMG I/Y’s 5' region.  This manuscript describes in detail the strategy used to sequence this promoter.

 Methods and Results

DNA extraction

One immature pea pod (3 cm length) from Pisum sativum was crushed between two sheets of weighing paper using pliers.  The pod was then transferred to a 1.5 ml microfuge tube with 1 ml of extraction buffer (6) (100 mM Tris, pH 8.0, 50 mM EDTA, 500 mM NaCl, 10 mM 2-mercaptoethanol).  140 µl of 10% SDS was added.  The tube was inverted to mix and incubated at 65 C for 10 minutes.  250 µl of 8M KOAc was added to the tube, inverted to mix, and placed on ice for 5 minutes.  The tube was then centrifuged at 13,000 rpm for 8 minutes and 600 µl of the supernatant was transferred to a new tube.  300 µl of isopropanol was added and the contents were mixed and held at 4 C for 10 minutes.  Following 10 minutes of centrifugation at 13,000 rpm the supernatant was discarded.  750 µl of 75% EtOH was added.  The tube was gently mixed then centrifuged for 3 minutes.  All supernatant was removed from the tube and the pellet resuspended in 50ul sterile ddH20.

 

Inverse PCR

      Pea genomic DNA was cleaved with the TaqI 4 bp cutter (BRL products/Life Technology, Grand Island , NY ) in the following 100 µl reaction mix: 10µl~500 ng DNA, 10 µl TaqI Buffer 2 (BRL), 5 µl TaqI, and 75 µl water.  Aliquots of the mix were incubated at 65° C for 30 min, 60 min, or 2 h.  TaqI had the specificity to cleave the TaqI site located within the 3' end of HMG I/Y gene open reading frame and various sites upstream or down-stream of the gene of interest. The digested DNA was purified with a phenol/chloroform extraction and EtOH precipitation. The DNA was resuspended in 100 µl water. Ten µl of this DNA solution was ligated to itself (using T4 DNA ligase, BRL) to create a series of small circular DNAs representing the total genome, including the HMG-I/Y gene and its adjacent sequence.  A primary PCR was performed as follows using the primer set indicated in Figs. 1 and 2:

Ligated TaqI/cut genomic DNA                                                         1.2 ul

10X PCR buffer (BRL)                                                                      2.2 µl

2.5 mM dNTPs                                                                                  1.2 µl

50 mM MgCl2                                                                                    0.8 µl

20 µm LeeInv367c (5’ GAA CAA CCG AAT GGC CTT CT 3’)          0.6 µl

20 µm LeeInv580F (5’ CCA AAG GCT TCT GGA AGT GG 3’)          0.6 µl

double distilled H2O                                                                           13.6 µl

Taq Polymerase                                                                                  0.2 µl

 

The PCR temperature  recycling program was: 94 C for 30 sec, 65 C for 20 sec, 72 C 1.5 min for 50 cycles.  The product was then diluted 1:10 in preparation for secondary PCR.

A secondary PCR utilized the primer combination INV615F (5' GCC GAA GAA GAT TGC TAG GAC 3')/INV248c (5' TCA TTC AGT GAA TCA ATA GCC 3') as indicated in Fig. 1 with an annealing temperature of 61 C.  This yielded an ~780 bp segment, which was cloned into a Topo PCR2.1 vector (Promega) and subsequently sequenced.  The two new sequences generated from the clone were recognized as being within the region 5' of the HMG I/Y start site. The length of this sequence (780 bp) was insufficient for the complete analysis of a HMG I/Y 5' region but contained an AluI site.  Thus the pea DNA was cut with AluI and the DNA was again circularized by ligation to obtain additional upstream sequence.  Two new sets of primers were designed.  Inv339F (5' ATC CTC ATC CAA AAG AAG 3’)/ Inv292 (5' AAT TAA GGC TTT TTT GAC 3') was used for the primary PCR reaction.   PCR was run under the following conditions; 94 C for 30 sec, 51 C for 20 sec, 72 C for 1 min.  Secondary PCR using Inv187c (5' TAA TTG AAA AGG GTA TGC 3')/Inc609f (5' TCA ATC CTT AGT TCA TCC 3') was then performed (94 C for 30 sec, 51 C for 20 sec, 72 C for 1 min for 50 cycles).  The subsequent PCR product was cloned and sequenced as before and increased the total sequence upstream of the HMG-I/Y gene to 1748bp (Fig. 1).

To verify the sequence 1748bp upstream of the HMG-I/Y gene, a primer set 13f (5’ ACA GAT TAT GAA CAA AGT TTA ACG 3’)/754 (5’ TCA CTT GTG TCA ACT GAG GC 3’) was used to amplify an approximate 2500 bp segment off of uncut genomic DNA.  PCR conditions were 94 C 30sec, 61 C 20sec, 72 C 2:4 min for 50 cycles.  The PCR product was cloned and completely sequenced to confirm that the inverse sequence reactions were assembled correctly.

Discussion

The availability of this sequence enables the identification of potential promoter elements 5' of the HMG-I/Y opening reading frame and assists the development of nested series of promoter/reporter elements to evaluate those sequences vital for activation (1).  Once these regulatory elements have been found it is possible to identify and characterize the sequences involved in 

HMG-I/Y transcription.  Stretches of alternating A and T sequences are known to bind the AT hooks of the HMG-I/Y protein (7).  There are fifteen 4 bp stretches, three 5 bp stretches, four 6 bp stretches and one 7 bp stretch of alternating A and T found in the region 5' of the pea HMG-I/Y gene open reading frame.  The availability of pure HMG-I/Y protein and the 5' sequence information enables gel mobility assays. These assays can determine if any of the regions 5' of HMG-I/Y associate with this architectural transcription factor that is its own coded gene product.  Many other predicted transcription factor binding sites can be derived from the 5' sequence.  For example, the MYB attachment site, AACCG, is found twice in the pea 5' region and once in the Arabidopsis promoter region (3).

 Acknowledgment: The GenBank Accession number for the pea HMG-I/Y sequence reported in this manuscript is AY864056. We thank the Washington Sea Grant program for support.  Ag. Research Center ms. no.0399.

1.  Choi, J.J., Klosterman, S.J. and Hadwiger, L.A.  2004.  Phytopath. 94: 651-660.

 2.  Gupta, R., Webster, C.I. and Gray, J.C.  1997.  Plant Mol. Biol. 35: 987-992.

3.  Gupta, R., Webster, C.I. and Gray, J.C.  1998.  Plant Mol. Biol. 36: 897-907.

4.  Klosterman, S. J., Choi, J. J.,Hadwiger, L. A.  2003.  Mol. Plant Path. 4: 249-258.

5.  Klosterman, S.J. and Hadwiger, L.A.   2002.  Plant Sci. 162: 855-866.

6.  Presting, G.G., Smith, O.P. and Brown, C.R.  1995.  Phytopath. 85:436-442.

7.  Reeves, R.  2001.  Gene 277: 63-81.

 

Text Box: Alu1 AGCTTCATTGATGTATACAGATTATGAACAAAGTTTAACGGAAAATTCAAGATTAGTTGA TCGAAGTAACTACATATGTCTAATACTTGTTTCAAATTGCCTTTTAAGTTCTAATCAACT 10 20 30 40 50 60 TTTCTTAATAAGCAATTTGTGATAAAATGAATTGAAAAATAAAACGAGTATAACTAATCA AAAGAATTATTCGTTAAACACTATTTTACTTAACTTTTTATTTTGCTCATATTGATTAGT 70 80 90 100 110 120 TGTATGCATGTGGATATATTAGTAGCAACCTAACCCTAACATCTCTTTATCATCTAACAT ACATACGTACACCTATATAATCATCGTTGGATTGGGATTGTAGAGAAATAGTAGATTGTA 130 140 150 160 170 180 CTTTGTTATTCTCTTATTTTAATTTGTTATTGTTTTAATTTCTCAAACAAAACATCTTTC GAAACAATAAGAGAATAAAATTAAACAATAACAAAATTAAAGAGTTTGTTTTGTAGAAAG 190 200 210 220 230 240 AAAACAAATCCTAAATTGCTTAAAATAGTAACAATTACTTCAACAATAAAAAAACTTTTA TTTTGTTTAGGATTTAACGAATTTTATCATTGTTAATGAAGTTGTTATTTTTTTGAAAAT 250 260 270 280 290 300 TTTCAATCATATTTTGTACATGCATCTCATTACATCTTGAAATTCCACTTATTTTCTGTA AAAGTTAGTATAAAACATGTACGTAGAGTAATGTAGAACTTTAAGGTGAATAAAAGACAT 310 320 330 340 350 360 CATGCATCTCATTACATTTTGAAATTCCACTTATTTTCTGGACATGGATCTCATTACATT GTACGTAGAGTAATGTAAAACTTTAAGGTGAATAAAAGACCTGTACCTAGAGTAATGTAA 370 380 390 400 410 420 TTGAAATTCCACTTATTTTCCGACAATTATTTTAAATTACTTAAAATAGTAACAATTACT AACTTTAAGGTGAATAAAAGGCTGTTAATAAAATTTAATGAATTTTATCATTGTTAATGA 430 440 450 460 470 480 CCAACAATTTTTTTTAATTCTATCATATTTTATACATACATTTTATTACATCTTGAAATT GGTTGTTAAAAAAAATTAAGATAGTATAAAATATGTATGTAAAATAATGTAGAACTTTAA 490 500 510 520 530 540 TCACTTATTCTCTAACAATTATCTTAAATTACTTAAAATAGTAATAATTATCTATAAATT AGTGAATAAGAGATTGTTAATAGAATTTAATGAATTTTATCATTATTAATAGATATTTAA 550 560 570 580 590 600 GTATCGTAAGATGATAAAAACATACTAACGAATTGTAGTAGTTTATAACTTAATATTTTT CATAGCATTCTACTATTTTTGTATGATTGCTTAACATCATCAAATATTGAATTATAAAAA 610 620 630 640 650 660 Taq1 TCTTTCGATTTTACTTTTATTATCTTAATTCAAAAAATTATATATTATTTAAATATATTT AGAAAGCTAAAATGAAAATAATAGAATTAAGTTTTTTAATATATAATAAATTTATATAAA 670 680 690 700 710 720 TTAAGTCATTTTATAATTATAAGTCATTTCATTTTATTCAACATTACAAATTTAATCAAT AATTCAGTAAAATATTAATATTCAGTAAAGTAAAATAAGTTGTAATGTTTAAATTAGTTA 730 740 750 760 770 780 TAATTTATTTTCAACCACCCATTACCAACTTATAAATTAAAAATAAATAAATTCATCAAC ATTAAATAAAAGTTGGTGGGTAATGGTTGAATATTTAATTTTTATTTATTTAAGTAGTTG 790 800 810 820 830 840 TATAAATTAATTTATCAATTATCCGTTATTTCTTTTAACAACAATGTCTATATACATATC ATATTTAATTAAATAGTTAATAGGCAATAAAGAAAATTGTTGTTACAGATATATGTATAG 850 860 870 880 890 900 GTATTAAAAATGATGATATAATTTCCACTTTTGATTTTTTAAAATCAAATTATGCAAAAA CATAATTTTTACTACTATATTAAAGGTGAAAACTAAAAAATTTTAGTTTAATACGTTTTT 910 920 930 940 950 960 Taq1 TATTTAAGAGGTCGGTGCGTCCCGACACTTAATATTTAGTATGAAAATTGTAATTATCGA ATAAATTCTCCAGCCACGCAGGGCTGTGAATTATAAATCATACTTTTAACATTAATAGCT 970 980 990 1000 1010 1020 AAATATACAAACCGAGTCAAACCGTTTCTTATTTTAGCAATAAAATTCACAGATACATTT TTTATATGTTTGGCTCAGTTTGGCAAAGAATAAAATCGTTATTTTAAGTGTCTATGTAAA 1030 1040 1050 1060 1070 1080 Text Box: ATCATTTTATTTTCCTATTGAATAAAATACAATGTTTTTTCATTTATTTAATCTTTAAAT TAGTAAAATAAAAGGATAACTTATTTTATGTTACAAAAAAGTAAATAAATTAGAAATTTA 1090 1100 1110 1120 1130 1140 Inv187c AATTTTTCTTGTTTATTTTATCACATTTTGATAACTATGAATTTGAAAAGCATACCCTTT TTAAAAAGAACAAATAAAATAGTGTAAAACTATTGATACTTAAACTTTTCGTATGGGAAA 1200 TCAATTAAAAAATCAATTTATTTATTTCATTTCATAAATAATATTCATAAAATTAAATAC AGTTAATTTTTTAGTTAAATAAATAAAGTAAAGTATTTATTATAAGTATTTTAATTTATG 1210 1220 1230 1240 1250 1260 Inv2923c AATGAGTAGAATTTCAAACTCTCAATAAATTTTAGTCAAAAAAGCCTTAATTTAAAAATA TTACTCATCTTAAAGTTTGAGAGTTATTTAAAATCAGTTTTTTCGGAATTAAATTTTTAT 1270 1280 1290 1300 1310 1320 Inv339f AATAAAAATATTTAAAATTGAGATAGTCTACATCACAAATCCTCATCCAAAAGAACAAAG TTATTTTTATAAATTTTAACTCTATCAGATGTAGTGTTTAGGAGTAGGTTTTCTTGTTTC 1330 1340 1350 1360 1370 1380 AATACAAAAAACAGTAGGTACCTCCAAATATTTCTGTGAACTAACACATTTTTGCCATGT TTATGTTTTTTGTCATCCATGGAGGTTTATAAAGACACTTGATTGTGTAAAAACGGTACA 1390 1400 1410 1420 1430 1440 CATCAATCCATGTGAGATTCTCCATATTATAATATCAACCCTTGGATCATCATCATTCTA GTAGTTAGGTACACTCTAAGAGGTATAATATTATAGTTGGGAACCTAGTAGTAGTAAGAT 1450 1460 1470 1480 1490 1500 TTGATTCCTAGCCGTCCATTGTCTTGTTCAGACAAACACAAGATATATCTTGGGAAAAAG AACTAAGGATCGGCAGGTAACAGAACAAGTCTGTTTGTGTTCTATATAGAACCCTTTTTC 1510 1520 1530 1540 1550 1560 AAGAGCAAACTTTTTTTAATATATTTAATTTCTTTCCAATCTTTTAATACATTTATCTCC TTCTCGTTTGAAAAAAATTATATAAATTAAAGAAAGGTTAGAAAATTATGTAAATAGAGG 1570 1580 1590 1600 1610 1620 Inv609F CTTTAAATTCAATCCTTAGTTCATCCATCAATTCACTCACAATCTCATTTCTCATAACAA GAAATTTAAGTTAGGAATCAAGTAGGTAGTTAAGTGAGTGTTAGAGTAAAGAGTATTGTT 1630 1640 1650 1660 1670 1680 AATTTCTATCTCCCTCAGATTTTTTATCTCAATTTTAAAGCTTTTTTCCTCACTCTTTCG TTAAAGATAGAGGGAGTCTAAAAAATAGAGTTAAAATTTCGAAAAAAGGAGTGAGAAAGC 1690 1700 1710 Alu1 1730 1740 Alu1 ORF HMG1F CAGCTTCAATGGCAACAAGAGAGGTTAATAAGCCTCTGTCACTTCCTCCTTACCCTGAGG GTCGAAGTTACCGTTGTTCTCTCCAATTATTCGGAGACAGTGAAGGAGGAATGGGACTCC 1750 1760 1770 1780 1790 1800 TAAACACAAACCCCAATTTTTACTTTTCTCATGGATATTTATTCTGTACTATTTTCTTAG ATTTGTGTTTGGGGTTAAAAATGAAAAGAGTACCTATAAATAAGACATGATAAAAGAATC 1810 1820 1830 1840 1850 1860 TAAAGTTGAAATTTTTTCACTGATCTGTTTGATCTCAAAATTTTCACTTACTTAGTTTTT ATTTCAACTTTAAAAAAGTGACTAGACAAACTAGAGTTTTAAAAGTGAATGAATCAAAAA 1870 1880 1890 1900 1910 1920 TTTTTCTCATCAAGTTTTGTTTGTTTTGGGTTTTTGTGTTTGTTTTGGGTTTTTGTTATT AAAAAGAGTAGTTCAAAACAAACAAAACCCAAAAACACAAACAAAACCCAAAAACAATAA 1930 1940 1950 1960 1970 1980 TTGATGGAAAAGATTGATTATGTGTTTTGTTGCATGTTTTTGTTGTAGTTGATACTGAAG AACTACCTTTTCTAACTAATACACAAAACAACGTACAAAAACAACATCAACTATGACTTC 1990 2000 2010 2020 2030 2040 Inv248c GCTATTGATTCACTGAATGAACCAAATGGATCAAACAAATCAGCAATATCAAACTACATA CGATAACTAAGTGACTTACTTGGTTTACCTAGTTTGTTTAGTCGTTATAGTTTGATGTAT 2050 2060 2070 2080 2090 2100 Inv367c GAATCAGTTTACGGTGAACTACCAGAAGGCCATTCGGTTGTTCTTTTATATCATCTGAAC CTTAGTCAAATGCCACTTGATGGTCTTCCGGTAAGCCAACAAGAAAATATAGTAGACTTG 2110 2120 2130 2140 2150 2160

Text Box: CAGATGAAAGAGAGTGGGGACCTTGTTTTTGCAAAGAACAACTACTTGAGGCCTGATCCA GTCTACTTTCTCTCACCCCTGGAACAAAAACGTTTCTTGTTGATGAACTCCGGACTAGGT 2170 2180 2190 2200 2210 2220 AATGCTCCACCGAAGAGAGGGCGCGGTAGGCCTCCTAAGGCGAAGGATCCGTTGGCCTCA TTACGAGGTGGCTTCTCTCCCGCGCCATCCGGAGGATTCCGCTTCCTAGGCAACCGGAGT 2230 2240 2250 2260 2270 2280 CCGCCTTCAGGTGCTGTGTCCACACCGAGGCCAAGGGGTCGTCCGCCTAAGGATCCTAAT GGCGGAAGTCCACGACACAGGTGTGGCTCCGGTTCCCCAGCAGGCGGATTCCTAGGATTA 2290 2300 2310 2320 2330 2340 Inv580F Inv 615F GCGCCACCGAAGACTCCAAAGGCTTCTGGAAGTGGTAGGCCAAGGGGTAGGCCGAAGAAG CGCGGTGGCTTCTGAGGTTTCCGAAGACCTTCACCATCCGGTTCCCCATCCGGCTTCTTC 2350 2360 2370 2380 2390 2400 ATTGCTAGGACCGAGGATGTTGATGCTTCAACTCCTAGTCCTGTGAGTGTTGCTGCTGTT TAACGATCCTGGCTCCTACAACTACGAAGTTGAGGATCAGGACACTCACAACGACGACAA 2410 2420 2430 2440 2450 2460 Taq1 AATGTTGATGTTGTTGTTCCATGTGTTGCTGCTGTTCCTACTTCGAGTGGGAGACCAAGG TTACAACTACAACAACAAGGTACACAACGACGACAAGGATGAAGCTCACCCTCTGGTTCC 2470 2480 2490 2500 2510 2520 GGTAGGCCTCCTAAGGTGAAGCCTCAGTTGACACAAGTGA CCATCCGGAGGATTCCACTTCGGAGTCAACTGTGTTCACT HMG754 2530 2540 2550 2560

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 1:  DNA sequence of the 5' region and the open reading frame of the pea gene HMG-I/Y. The restriction enzyme sequences and the primers utilized in developing the 5' sequence are underlined and labeled.

 

 

Fig. 2  Cartoon of the sequence of restriction digestion, primer development and DNA sequencing analyses of the pea HMG-I/Y 5' region (See Methods).