ZmDB Title logo

Comparison of RNA expression profiles based on maize EST frequency analysis and microarray hybridization  (supplemental information)

John Fernandes, Volker Brendel, Xiaowu Gai, Shailesh Lal, Vicki L. Chandler, Rangasamy Elumalai, David W. Galbraith, Elizabeth Pierson, and Virginia Walbot

Abstract

Assembly of 73,000 Expressed Sequence Tags (ESTs) representing multiple organs and developmental stages of Zea mays L. identified ~22,000 tentative unique genes (TUGs) at the criterion of 95% identity. Based on sequence similarity, overlap between any two of nine libraries with more than 3,000 ESTs ranged from 4-20% of the constituent TUGs. The most abundant ESTs were recovered from only one or a minority of the libraries, and only 26 EST contigs had members from all nine EST sets (presumably representing ubiquitously expressed genes). For several examples, ESTs for different members of gene families were detected in distinct organs. To study this further, two types of microarray slides were fabricated, one containing 5,534 ESTs from 10-14 day endosperm, and the other 4,844 ESTs from immature ear, estimated to represent about 2,800 and 2,500 unique genes, respectively. Each array type was hybridized with fluorescent cDNA targets prepared from endosperm and immature ear polyA+ RNA. Although the 10-14 day post-pollination endosperm TUGs showed only 12% overlap with immature ear TUGs, endosperm target hybridized with 94% of the ear TUGs, and ear target hybridized with 57% of the endosperm TUGs. Incomplete EST sampling of low abundance transcripts contributes to an underestimate of shared gene expression profiles. Reassembly of ESTs at the criterion of 90% identity suggests how cross-hybridization among gene family members can overestimate the overlap in genes expressed in microarray hybridization experiments.

 

Supplemental information to this article, published in Plant Physiology, is provided on this web page.

 

Tables

Table II (Expanded).  Number of ESTs by library showing TUC composition

Table IV.  Abundant ESTs with protein matches and number of contributing sources

Table V.  TUCs representing constitutively expressed genes

Table VI.  Microarray controls

 

Doughnut Charts Showing TUG Overlap of a Library to all Other Libraries

Library 486     Library 605     Library 606

Library 614     Library 618     Library 660

Library 687     Library 707     Library 946

(See the Library 486 chart for identification of the ring order)

Pie Charts Showing TUG Overlap Between Early Libraries (486, 603, 605, 606, 614)

http://gremlin3.zool.iastate.edu/zmdb/EST/reports.html

 

Table II (Expanded).  Number of ESTs by library showing TUC composition

Number of ESTs in each library for the set of TUCs which contain ESTs from a certain number of libraries.  Counts are based on the 9/27/2000 assembly at ZmDb.  See below for library descriptions.

No. of Libraries

TUCs (N)

486 ESTs

605 ESTs

606 ESTs

614 ESTs

618 ESTs

660 ESTs

687 ESTs

707 ESTs

946 ESTs

1

11280

1708

1519

1180

2319

858

1328

1213

2575

1638

2

3133

903

1146

891

1541

654

1181

664

1428

1035

3

1353

631

1045

543

1059

534

566

526

696

875

4

628

467

654

523

609

349

362

347

392

770

5

323

313

522

364

411

255

298

244

270

678

6

191

287

436

408

372

226

220

226

226

577

7

111

295

418

414

225

154

183

267

271

415

8

51

145

212

240

174

93

120

111

92

213

9

26

160

157

282

118

75

78

118

102

183

Total

17096

4909

6109

4845

6828

3198

4336

3716

6052

6384

 

Library Description

486

Leaf primordia

605

10-14 day endosperm

606

1-2 cm immature ear

614

4-day root

618

<2 cm tassel

660

Mixed tassel stages

687

Mixed embryo stages

707

Mixed adult organs

 

Table IV. Abundant ESTs with protein matches and number of contributing sources

TUCs with at least 50 ESTs and high sequence similarity to a known protein are displayed. An additional four TUCs of more than 50 ESTs had no match to known proteins.

TUC

Number of ESTs

Number of sources

Gene product

TUC09-07-7674.1

172

9

α-tubulin 1

TUC04-05-9207.2

132

2

Zein Zc2

TUC09-07-7412.1

126

7

Glycine-rich protein

TUC07-14-6708.1

106

3

Zein 19 kDa protein

TUC09-07-5159.1

101

8

S-adenosyl-L-homocysteine hydrolase

TUC09-07-7463.1

94

8

Ribosomal protein S8

TUC09-07-7768.1

91

9

S-adenosylmethionine decarboxylase

TUC09-07-7895.1

88

9

MNB1b mRNA for DNA-binding protein

TUC07-14-6710.1

87

3

Zein Zd1

TUC09-07-7584.1

79

7

Cyclophilin

TUC09-07-7228.1

77

9

Cytochrome P450 monooxygenase

TUC09-07-7689.1

69

9

Elongation factor 1α

TUC09-07-7800.1

65

7

α-tubulin 3

TUC09-07-7470.1

64

5

H2B histone

TUC09-07-7473.1

63

9

A. thaliana - MYH19

TUC09-07-7633.1

61

6

H2A histone

TUC09-07-7564.1

59

9

Elongation factor 1β

TUC09-07-5230.1

57

9

α-tubulin 2

TUC09-07-7544.1

55

7

H1 histone   

TUC09-07-7491.1

54

8

Elongation factor 2

TUC09-07-6854.2

53

9

Ribosomal protein L10

TUC09-07-5525.1

51

7

Aldolase

TUC09-07-7857.1

50

7

Hsp70-5 heat shock protein

 

 

Table V. TUCs representing constitutively expressed genes

The table lists TUCs consisting of ESTs from all nine sources. Gene products were inferred on the basis of high sequence similarity. Not shown: five contigs encoding ribosomal proteins, three contigs without protein matches.

 

Number of Sequences in Each Library

 

Gene Product

486

605

606

614

618

660

687

707

946

Total

TUC ID (from 9/7/2000 assembly)

α-tubulin 1

 

11

 

4

 

118

 

2

 

2

 

9

 

9

 

5

 

12

 

172

TUC09-07-7674.1

α-tubulin 2

5

5

23

3

2

7

3

8

1

57

TUC09-07-5230.1

MNB1b-like DNA-binding protein

10

4

1

3

11

2

7

8

42

88

TUC09-07-7895.1

Transcriptional coactivator-like protein

2

2

1

5

3

3

1

1

5

23

TUC09-07-6649.1

Eukaryotic translation initiation factor 6

1

2

2

4

3

1

2

2

3

20

TUC09-07-5880.1

Elongation factor 1 α

10

11

8

10

6

6

8

3

7

69

TUC09-07-7689.1

Elongation factor 1 α

8

8

4

4

1

5

9

1

3

43

TUC09-07-4536.2

Elongation factor 1 β

10

11

11

4

2

1

5

3

12

59

TUC09-07-7564.1

Elongation factor 1B γ

5

3

1

2

2

3

3

3

1

23

TUC09-07-309.1

Aldolase

4

13

6

1

1

2

9

4

1

41

TUC09-07-6714.1

Cytochrome P450 monooxygenase

5

8

14

1

1

2

8

25

13

77

TUC09-07-7228.1

Enolase (eno2)

7

1

11

5

6

5

10

3

2

50

TUC09-07-7631.1

Gluthathione S-transferase I

8

2

1

20

3

3

1

3

5

46

TUC09-07-7038.1

S-adenosylmethionine decarboxylase

15

2

33

4

1

3

16

10

7

91

TUC09-07-7768.1

S-adenosyl-L-methionine synthetase

4

1

7

6

1

3

1

1

1

25

TUC09-07-3386.1

Triticum aestivum heat shock protein

3

2

1

5

1

1

1

1

1

16

TUC09-07-3948.1

A. thaliana - MYH19

6

10

5

4

10

1

4

1

22

63

TUC09-07-7473.1

Mitochondrial F-1-ATPase subunit-2

3

1

2

1

1

2

4

2

2

18

TUC09-07-7917.1

 

Table VI. Microarray Controls

To normalize signal intensities, we applied rank correlation analysis to identify a subset of the plant control genes whose expression patterns were similar among tissues.   These controls were printed on slides 605.03, 605.04, 606.01, and 614.01.

Sequence Description

Sequence Designation

Vector

Lab of Origin

PCR Product Size (kb)

GENE ubiquitin2 (skuqbgii cDNA)

vc9

Bluescript II SK+

Chandler lab

0.4

GENE ubiquitin2 (ubiquitin2 975 bp psti)

vc18

Bluescript II SK+

Chandler lab

1.2

Maize GST I cDNA

pBS- GST I

Bluescript II SK-

Walbot lab

1.15

Maize GST III cDNA

pBS- GST III

Bluescript II SK-

Walbot lab

1.2

Arath act 2/7, actin 11

605053G09

TC 8935

Galbraith lab

1.5

Actin 11

605017E10

TC9076

Galbraith lab

1.5, 0.7

EF1A

605077A07

TC5642

Galbraith lab

1.3

EF1A

605057B12

TC5644

Galbraith lab

0.4

EF1A

605017C01

TC5648

Galbraith lab

1.6

Elongation Factor TU

605065A08

TC1173

Galbraith lab

1.5

Elongation Factor TU

605010F02

TC1173

Galbraith lab

0.7

Elongation Factor TU

687044D07

TC8092

Galbraith lab

1.7

Elongation Factor TU

605019C05

Galbraith lab

1

Glutamine synthetase

603029G03

TC7408

Galbraith lab

1.7

Glutamine synthetase

603006F12

Galbraith lab

1

Histone H1 homolog

605054D10

TC9311

Galbraith lab

0.7

Histone H2A homolog

605058D08

TC9490

Galbraith lab

0.8

Histone H2A homolog

605065C09

TC8776

Galbraith lab

0.8

Histone H2A homolog

605088B07

Galbraith lab

0.7

Histone H4

605004D02

TC8789

Galbraith lab

0.7

HMG1/2 like protein

606045H01

TC7929

Galbraith lab

1

HMG1/2 like protein

606055D02

Galbraith lab

1

MNBB DNA binding protein

605070E11

TC8049

Galbraith lab

1.1

MNBB DNA binding protein

618049A09

Galbraith lab

0.5

Tubulin alpha 1/2/3 chain

605072H11

TC8857

Galbraith lab

1

Tubulin alpha ½ chain

605032H06

TC8859

Galbraith lab

1.5

Tubulin alpha ½ chain

605054G06

TC8858

Galbraith lab

1.6

Tubulin alpha 3 chain

605028B02

TC9236

Galbraith lab

1.5

Vacuolar ATPase 69Kda subunit (A)

605077C08

TC5836

Galbraith lab

1.7

 

Full list of microarry controls printed on each slide

http://gremlin3.zool.iastate.edu/zmdb/microarray/controls.html

 

Doughnut Charts