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Proc Natl Acad Sci U S A. 2002 Jan 22; 99(2): 850–855.
Published online 2002 Jan 15. doi:  10.1073/pnas.022627999
PMCID: PMC117394

Physical and genetic mapping of barley (Hordeum vulgare) germin-like cDNAs


Germin with oxalate oxidase and superoxide dismutase activity is a homohexamer of six manganese-containing interlocked β-jellyroll monomers with extreme resistance to heat and proteolytic degradation [Woo, E.-J., Dunwell, J. M., Goodenough, P. W., Marvier, A. C. & Pickersill, R. W. (2000) Nat. Struct. Biol. 7, 1036–1038]. This structure is conserved in germin-like proteins (GLPs) with other enzymatic functions and characteristic for proteins deposited in plant cell walls in response to pathogen attack and abiotic stress. Comparative nucleotide and amino acid sequence analyses of 49,610 barley expressed sequence tags identified 124 germin and germin-like cDNAs, which distributed into five subfamilies designated HvGER-I to HvGER-V. Representative cDNAs for these subfamilies hybridized to 67 bacterial artificial chromosome (BAC) clones from a library containing 6.3 genomic equivalents. Twenty-six BAC clones hybridized to the subfamily IV probe and identified a gene-rich region including clone 418E1 of 96 kb encoding eight GLPs (i.e., 1 gene per 12 kb). This BAC clone lacked highly repeated sequences and mapped to the subtelomeric region of the long arm of chromosome 4(4H). Among the six genes of the contig expressed in leaves, one specifies a protein known to be associated with papilla formation in the epidermis upon powdery mildew infection. Three structural genes for oxalate oxidase are present in subfamily I and eight GLPs of various functions in the other subfamilies. These genes map at loci in chromosomes 1(7H), 2 (2H), 3(3H), 4(4H), and 7(5H). Some are present on a single BAC clone. The results are discussed in relation to cereal genome organization.

Barley germin is a 96-kDa homohexamer of six manganese-containing interlocked β-jellyroll monomers with extreme resistance to heat and proteolytic degradation. It functions as a hydrogen peroxide-generating oxalate oxidase (OXO; EC and displays manganese superoxide dismutase (MnSOD) activity (13). Germin was discovered as a protein synthesized during cereal grain germination (4). Remodeling of the plant cell walls during pathogen attack or abiotic stress is associated with the expression of germin or germin-like proteins (GLPs), which may lack detectable OXO activity (reviewed in ref. 5). Germins are functionally diverse but structurally related members of the cupin superfamily, including isomerases, cyclases, dioxygenases, proteins binding sugars, or auxin and monomeric germin dimer globulin seed storage proteins such as phaseolin (5, 6). MnSOD activity was demonstrated for GLPs of the moss Barbula unguiculata (7) and tobacco nectarin, a defense protein of floral reproductive tissues (8). ADP glucose pyrophosphatase/phosphodiesterases (AGPPase) from barley leaves are oligomeric GLPs (9). OXOs and GLPs have been isolated from wheat (10), barley (1, 2), maize, oat, rice, and rye (11). They are synthesized with a presequence of 23 or 24 aa and targeted into the cell wall and apoplast (1, 12). In contrast to advanced knowledge of the structural, cell biological, and expression features of barley and wheat germins and GLPs, less is known about their genomic organization. Germin and GLP diversity has been probed with Arabidopsis thaliana-expressed sequence tag (EST) libraries (13, 14). Limited linkage mapping data are available for wheat germin (15), and copy numbers have been estimated for several GLPs with Southern blots and differential hybridizations (16, 17). Elucidation of the genomic organization of the GLP family in cereals is a prerequisite for identifying the functions of the individual family members and their utilization in breeding of cereal crops. Examples of the utility of these genes are suppression of powdery mildew infection by transient expression of individual GLPs with a constitutive promoter (18) and utilization of individual GLP gene promoters for tissue-specific expression of heterologous genes (12, 19). Here we present an analysis of 173 barley GLP sequences found among the nearly 50,000 sequenced and annotated barley EST clones. According to their amino acid sequence homology they fall into five subfamilies designated HvGER-I to HvGER-V. Representative members for each family were used for physical and genetic linkage mapping.

Materials and Methods

Sequence Analysis of Germin and Germin-Like ESTs.

The EST cDNA sequences were downloaded from the Clemson University Genomics Institute Barley EST Database (http://www.genome.clemson.edu/projects/barley). Approximately 5,000 clones were sequenced from each of 10 libraries that are described on the Clemson web page. A pairwise similarity matrix of the nucleotide sequences was generated by using the genetool alignment program (Double Twist, Oakland, CA). Sequences with >80% similarity were divided into 12 groups (see Table 3, which is published as supporting information on the PNAS web site, www.pnas.org). The netblast program, available from the National Center for Biotechnology Information home page at http://www.ncbi.nlm.nih.gov, was used for blastn and blastx analyses. blastn results were manually inspected, and corrections of the groupings were performed by joining sets of sequences with >85% similarity spanning >150 bp into one of five groups. Representative clones from each group were selected, confirmed by sequencing, and used as probes for physical and genetic mapping. Assignments of all barley germin-like EST entries to subfamilies are included in Table 3.

Bacterial Artificial Chromosome (BAC) Library Analysis.

A barley cv. Morex BAC library made in pBeloBAC11 vector (20, 21) was screened with 32P-labeled probes. BAC DNA from positive colonies was isolated by using a standard alkaline lysis procedure (22). Southern blot analysis for clone identification and fingerprinting was performed on HindIII-digested BAC DNA using separate 32P-labeled probes.

Southern Blot of Total Barley Genomic DNA.

DNA isolation, Southern blotting, and hybridization have been described (23). Hybridization was at 65°C followed by washing at 65°C in a solution containing 2× SSC with 1% SDS for 20 min, 1× SSC with 1% SDS for 20 min, 0.5× SSC with 1% SDS for 20 min, and optionally 0.2× SSC with 1% SDS for 20 min.

Pulsed-Field Gel Electrophoresis (PFGE) of BAC Clones.

PFGE was performed with the CHEF-DR III system (Bio-Rad) according to the manufacturer's instructions. Essentially, 200 ng of restriction enzyme-digested BAC DNA was loaded and PFGE was performed for 15 h at 6 V with 1- to 6-s switch time. Low Range PFG Molecular Weight Marker (New England Biolabs) was the molecular size standard.

Fingerprinting of HvGER-IV BAC Clones.

Contigs were assembled from autoradiographs of HindIII-digested BAC DNA hybridized with the BE216336 clone. Hybridizing bands were manually compared in pairs, eliminating “buried” bands. BAC insert sizes were determined from NotI-digested BAC DNA separated by PFGE. Alternatively, HindIII-digested BAC DNA and molecular size marker DNA was 32P-end-labeled, subjected to agarose gel electrophoresis, and Southern-blotted. The bands were visualized after 5 h exposure on x-ray film, scanned, and optimized by using bitmap editing software. The contig was assembled by elimination of buried bands.

Linkage Mapping.

Genetic mapping populations were: Steptoe × Morex, 150 doubled haploid lines (DHLs) (23); Harrington × Morex, 140 DHLs (24); Steptoe × Q21861 (144 DHLs) (A. Kleinhofs, unpublished work); Oregon Wolf Barley Dominant × Recessive (94 DHLs) (25); and Gorbernadora × CMB643 (144 DHLs) (26). Mapping data are at http://wheat.pw.usda.gov. Genetic distances were calculated with mapmaker and/or mapmanager software.

Construction of Gene-Specific Probes.

Restriction fragment length polymorphism (RFLP) probes representing parts of the noncoding regions of the genes were obtained by PCR using subclone templates (Table 4, which is published as supporting information on the PNAS web site). Alternatively, RFLP probes were generated directly from the BAC clone by subcloning HindIII-digested BAC DNA in pBluescript SK vector (Stratagene). Subclones (usually 96 per BAC clone) were negatively selected by using total barley DNA and GLP probes to obtain low-copy clones unrelated to the GLP sequence.


Sequence Analyses of Barley Germin-Like cDNAs Reveal Five Subfamilies.

We identified 173 GLP sequences among 49,610 sequenced and annotated barley EST clones. blast homology search and a two-by-two alignment matrix grouped the clones into five distinct subfamilies, HvGER-I to HvGER-V (Table (Table1).1). Representative members of each subfamily were isolated from barley cDNA libraries and confirmed by nucleotide sequencing. Tables Tables11 and and22 summarize the analyses of the EST sequences. More than half of the sequenced germin-like EST clones are members of the HvGER-I subfamily. The majority of the HvGER-I clones were obtained from root, stressed shoot, and leaf cDNA libraries. Among the 91 HvGER-I ESTs 74 had ORFs of sufficient length to permit meaningful in silico translation into amino acid sequences (compare Table 3). Discounting sequencing mistakes, 39 of the ORFs were identical at the nucleotide and amino acid level to the barley OXO HvOxOa cDNA (GenBank accession no. Y14203) and 30 matched the transcript of the OXO HvOxOb gene (GenBank accession no. U01963). They could be assigned unequivocally by the 18 aa differences distinguishing the two precursor proteins (16). Five ESTs with ORF identity were isolated from the two spike libraries. They had a sequence with 85% residue identity to OXO from barley and wheat. This third OXO gene may encode the enzyme strongly expressed in the developing epicarp (12).

Table 1
Key to the barley germin (GER) or GLP subfamilies
Table 2
Distribution of ESTs among germin-like subfamilies

Among the 35 HvGER-II cDNA clones, 28 originated from cold- or dehydration-stressed seedling shoots. Sixteen of these ESTs translated into ORFs with amino acid and nucleotide sequence identity to HvGLP encoding a soluble ADP glucose pyrophosphatase (GenBank accession no. Y15962). It is likely that all 35 ESTs originate from transcripts of the same gene. Eight of the 24 sequenced HvGER-III clones were isolated from the cDNA library of unstressed roots. Stressed shoots and immature spikes were also a major source for HvGER-III clones. Among these ESTs, 15 were unequivocally assignable to the GerB gene (GenBank accession no. AF250934). Amino acid 86 was consistently N and not T as reported previously (12). Ten of 12 HvGER-IV clones were obtained from powdery mildew-infected leaf cDNA libraries. Three of the ESTs of this group were assignable to the papilla-specific leaf GLP (GenBank accession no. X93171), whereas four ESTs indicated novel GLPs. Members of the HvGER-V subfamily were isolated primarily (10 of 11) from leaf and root cDNA libraries. A nucleotide sequence alignment of the 11 ESTs separated them into two groups, one with five members yielding a consensus sequence corresponding to GLP (GER7) of rice (GenBank accession no. AF072694) (28) and the other with six members corresponding to GER4 of rice (GenBank accession no. AF032974) (28).

No less than 68 of the 173 analyzed GLP cDNA clones derived from roots of etiolated seedlings, whereas 56 clones originated from shoots of seedlings either stressed by a cold or a dehydration treatment. Unstressed, dark-grown shoots yielded only two GLP cDNA clones, and no GLP cDNAs were found among 4,705 sequenced EST clones from a testa/pericarp library.

Physical Mapping of Barley GLPs by Probing a BAC Library.

Screening the BAC library with representatives of the subfamilies HvGER-I to HvGER-V yielded 67 clones (Table 5, which is published as supporting information on the PNAS web site). Two BAC clones hybridized strongly with the representative for subfamily I (BE214541), clone 1 with two different molecular-sized HindIII bands and clone 5 with a third different-sized band (Fig. (Fig.11A). This finding is in agreement with the transcripts of three genes identified in this family by the ESTs. Representative for subfamily V (BE216618) hybridized strongly to one band in clones 4 and 6 and to a different-sized band of clone 1 (Fig. (Fig.11E). The presence of additional GLP genes on the BAC clones is revealed by weakly hybridizing bands. Some of the bands hybridizing with different intensity to the subfamily I as well as subfamily V probe appear to have the same size and may be the same gene (e.g., Fig. Fig.1,1, clones 4 and 6). The two BAC clones hybridized to the subfamily II probe (Fig. (Fig.11B). The subfamily III cDNA clone, BE195143, hybridized to 58 BAC clones, all of which cross-hybridized with the subfamily IV probe (BE216336). This probe hybridized strongly to the BAC clones previously identified to carry the germin genes GerA and GerC and more weakly to the BAC clones containing germin genes GerB, GerD, GerF, and GerE (12) (Fig. (Fig.11 C and D). Germin genes GerB, GerD, GerF, and GerE appear to reside at separate loci because no overlapping BAC clones were found. Clones 7–11 of Fig. Fig.11 are typical for subfamily III and IV selected BAC clones. In agreement with previous data (12) the cDNA clone of subfamily III, BE195143, hybridizes less strongly to the HindIII fragments characteristic for the BAC clones selected with the subfamily IV probe (Fig. (Fig.11C). BAC clone 418E1 (Fig. (Fig.11D, clone 9) and some other subfamily IV BAC clones (see the next section) contain up to eight hybridizing bands, suggesting clustering of several GLP-encoding genes.

Fingerprinting of HvGER-IV BAC Clones Establishes a Gene-Rich Contig.

Twenty-six BAC clones hybridized strongly to the subfamily IV probe, BE216336. Most of them were previously identified as members of the GLP subfamilies containing genes GerA and GerC (12). Fingerprinting of the BAC clones 418E1 and 677H20 identified the same pattern with eight hybridizing bands (Fig. (Fig.22A). Restriction fragments 1, 3, 4, 6, or 7 hybridized in various combinations in other BAC clones. Furthermore, the 3.3-kb fragment in clones 617i14, 418E1, 677H20, and 705B5 is substituted for by a fragment with slightly higher mobility in clones 799E9, 671N8, and 670M13. This fragment with differential mobility is believed to arise from the same germin gene with a HindIII site that terminates some of the BAC clones, suggesting that the HvGER-IV contig is composed of nine hybridizing HindIII restriction fragments. BAC clones 418E1 and 677H20 contain an internal NotI site, dividing 418E1 into a 24-kb and a 72-kb fragment, but 677H20 into a 15-kb and 80-kb fragment (Fig. (Fig.22 B and C). Two NotI sites are in the vector flanking the insert. Hybridization was positive to all four fragments, but hybridization intensity suggests that most subfamily IV genes are located in the 72-kb fragment of clone 418E1. Clones 9D23, 275i3, 715L7, and 17D16 overlap clones 418E1, 617i14, and 677H20 (Fig. (Fig.22A) because they share three hybridizing fragments of the same size. Southern blot hybridization of the NotI-digested 9D23, 275i3, 715L7, and 17D16 BAC clones revealed two internal NotI sites. A positive hybridization signal of all three resulting fragments with the subfamily IV probe suggested that the contig extends beyond the BAC clones 418E1 and 799E9, two BAC clones with a single internal NotI site. Apparently the contig extension is a ≈45-kb NotI fragment present in 9D23, 275i3, 715L7, and 17D16, but absent from 418E1 and 799E9. At the other end the contig is flanked by BAC clones 376A4 and 545F14 with a size of 105 and 115 kb, respectively. They are characterized by a single germin-containing HindIII fragment of about 700 bp (Fig. (Fig.22A). There is an internal NotI site in both of these BAC clones, but only one of the resulting fragments (≈15 kb) hybridizes with the subfamily IV probe (Fig. (Fig.22C). This analysis leads to an estimate of 150 kb for the chromosome region housing the nine HvGER-IV subfamily genes (Fig. (Fig.22D).

Figure 2
Fingerprinting of HvGER-IV BAC clones. (A) Southern blot of HindIII-digested HvGER-IV BAC clones. Arrow indicates two slightly different-sized bands within 418E1 and 799E9 BAC clones. M, molecular size standard. (B) Ethidium bromide-stained PFGE image ...

Genetic Mapping of Barley GLPs.

Hybridization of the HvGER-I subfamily probe to restriction enzyme digested genomic DNA of cv. Morex resulted in three bands, indicating up to three genes, of which at least two are very closely linked physically, because they are present on the same BAC clone. These three genes are the OXO genes HvOxOa, HvOxOb, and a third gene identified by five ESTs. Mapping of the HvOxOa gene (subfamily I clone BE214541) in the Steptoe × Morex population identified a single locus, on chromosome 4(4H) bin2 (Fig. (Fig.3).3). The two bands present also in Steptoe cosegregated with the larger fragment in Morex. The other two restriction fragments were not polymorphic and could not be mapped. The weakly hybridizing bands that cross-hybridize with HvGER-V BAC clones (Fig. (Fig.11A) were not detected in genomic Southern blots. The subfamily II cDNA clone identifying the HvGLP gene encoding an ADPglucose pyrophosphatase was mapped to a single locus AW982932.B on chromosome 1(7H) bin1 (Fig. (Fig.3).3). However, a weakly hybridizing band in cv. Harrington mapped to a different locus, AW982932.A on chromosome 2(2H) bin5 (Fig. (Fig.3).3). This band was not seen in the BAC library screen and may be absent in cv. Morex. The HvGER-V probe identifying a GLP gene homologous to germin7 of rice was mapped to a single locus BE216618 on chromosome 7(5H) bin13 (Fig. (Fig.3).3). Using a combination of several mapping populations and restriction enzyme polymorphisms we mapped the HvGER-III probes ARD1776.1 (GerB) and ARD1771.1 (GerF) to two closely linked loci on chromosome 1(7H) bin8.

Figure 3
Barley Steptoe × Morex bin map with integrated GLPs (boxed in gray). HvGER subfamilies are indicated in parenthesis. GerA, GerB, GerC, GerD, GerE, and GerF are previously described (12) barley GLP genes. Some of the genes are not positioned precisely, ...

Probe ARD1769.1 (GerD) mapped to a locus on chromosome 3(3H) bin4, and probe ARD1772.1 (GerE gene) mapped to chromosome 7(5H) bin3. ARD1768.1 (GerA) is located on chromosome 4(4H) bin13 (Fig. (Fig.3).3). The GerA gene was suspected to be one of the nine genes from the germin-rich contig because it cosegregated with the HvGER-IV-derived probes ARD2848 and ARD2940 mapped to chromosome 4(4H) bin13. These probes were developed from BAC clones 418E1 (ARD2848) and 799E9 (ARD2940) to determine whether they mapped to the same locus. There was some concern about slightly different fingerprint patterns in one of the bands identified with the germin probe (Fig. (Fig.22A). The ARD2848 and ARD2940 loci cosegregated with one another and with the ARD1768.1 (GerA) locus, indicating that the nine-GLP gene cluster resides on chromosome 4(4H) bin13.


Among the 173 barley ESTs encoding germin or GLPs, 124 had sufficient length to assign them by nucleotide and deduced amino acid sequence identity/similarity to five subfamilies designated HvGER-I to HvGER-V. Subfamily HvGER-I comprised the two previously identified structural genes for barley OXO, HvOxOa and HvOxOb (17). Mapping of HvOxOa located it on the short arm of chromosome 4(4H), orthologue of wheat chromosomes 4A, 4B, and 4D, and thus consistent with the map position of a wheat OXO gene (15). Isolation of several ESTs of these two genes from the cDNA libraries of the root, from cold-stressed shoots and mildew-infected leaves, is in agreement with previously established increased expression of these enzymes upon fungal infection and environmental stress in barley (17, 29, 30) and wheat (1, 31). ESTs from the spike cDNA libraries identified a third, previously unknown, gene in this subfamily. Two of the three genes are closely linked because they are located on a single BAC clone.

The 35 ESTs of subfamily HvGER-II originate from the barley gene HvGLP1, which has been mapped in cv Morex to the distal region of chromosome 1(7H) bin1. The gene encodes an ADP glucose pyrophosphatase/phosphodiesterase, which is targeted to and insolubilized in the cell walls of barley (9, 27). A second isoform of this enzyme has been isolated from barley leaves (9) and may be encoded by the gene cross-hybridizing with the cv Harrington gene located on chromosome 2(2H) bin5. This gene seems to be lacking in cv Morex. In agreement with the observation that HvGLP1 is up-regulated upon biotic and abiotic stress, the majority of the ESTs originated from the library of dehydration-stressed shoots. Rice contains two GLPs with similar sequence characteristics, OsGER5 (28) and OsGLP1 (C. Yoshizawa, M. Ono, and M. Inoue, personal communication).

The barley GerB gene is specifically expressed in the testa (seed coat) of the developing grain (12). Accordingly the probe ARD1776.1 derived from the HvGER-III subfamily GerB gene identified six ESTs from the two spike libraries. It also identified four ESTs from the stressed-shoot library and four from the seedling roots. The testa tissue undergoes programmed cell death during grain development, a process also characteristic for tracheary element formation in root development and for senescence of a number of tissues. GerB may thus be involved in these processes. The gene was mapped to chromosome 1(7H) bin8 close to GerF (ARD1769.1). However, the two genes are not on the same BAC clone. Barley gene GerD (ARD1769.1) was mapped to chromosome 3(3H) bin4 and GerE (ARD1772.1) to chromosome 7(5H) bin3. These genes are related to wheat GLP 13.3 (32) and rice GLPs, OsGER1 and OsGER 2 (28). Subfamily HvGER-V ESTs show homology to the previously identified rice GLPs OsGER7 and OsGER4 (28).

Three of the ESTs of the HvGER-IV family were from the HvOxOLP gene. The protein specified by this gene accumulates in the epidermal tissues and is associated with papilla formation upon powdery mildew infection (16) in contrast to the mildew-induced structural gene for OXO HvOxOa expressed in the mesophyll cells (16, 17). The other four represented novel genes. The GerA gene, similar but not identical to the HvOxOLP, and the two HindIII subclones from BAC clones 418E1 and 376A4 of the contig containing nine GLP genes (Fig. (Fig.22D) mapped to chromosome 4(4H) bin13. This is a gene-rich region with eight of the genes in a 96-kb fragment or 1 gene per 12 kb. Gene-rich segments or gene spaces with clustered genes in ≈100 kb with a remarkable uniform base composition were first identified for barley, maize, and rice with CsCl density gradient profiles and interpreted to mean that differences in genome sizes were caused primarily by the amount of repetitive DNA separating the gene spaces (33). Sequencing of contiguous genomic stretches in maize (34, 35), sorghum (34), wheat, rice, and barley (3638) has verified that genome expansion and contraction commonly takes place by addition or removal of large blocks of retrotransposon elements and miniature inverted repeat transposable elements. In barley the BARE-1 retrotransposon is present in 16.6 × 103 copies and more than 6 × 104 single long terminal repeats (39). It has been further shown that genes can be scattered singly among retrotransposon blocks or can be organized in a retrotransposon-free space with as many as 10 genes (35). We have previously estimated the amount of repetitive sequences by separating HindIII-restricted BAC DNA fragments on agarose gels and hybridizing them with labeled total barley genomic DNA. The GerA-containing clones and the BAC clone 418E1 with the eight GLP genes contained only one strongly and one weakly hybridizing band, suggesting very few repeated DNA sequences (12). Multiple bands on BAC clone 376A4 indicated that this clone contains many repeated sequences (12). The GLP gene cluster identified here comprises nine functionally related genes. It will be interesting to determine whether this gene-rich contig contains other functionally unrelated genes and to determine its recombinogenic properties.

Supplementary Material

Supporting Tables:


This is Scientific Paper No. 0210–05 from the College of Agriculture and Home Economics Research Center, Washington State University, Project 0196. This research was supported by U.S. Department of Agriculture National Research Initiative Grant 99–35300–7694 and by the U.S. Department of Agriculture Cooperative State Research, Education and Extension Service/National Research Initiative Competitive Grants Program (Grant 9701248).


oxalate oxidase
germin-like protein
expressed sequence tag
bacterial artificial chromosome
pulsed-field gel electrophoresis
doubled haploid line


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    PubChem Compound links
  • EST
    Published EST sequences
  • Gene (nucleotide)
    Gene (nucleotide)
    Records in Gene identified from shared sequence links
  • MedGen
    Related information in MedGen
  • Nucleotide
    Published Nucleotide sequences
  • Protein
    Published protein sequences
  • PubMed
    PubMed citations for these articles
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    Related taxonomy entry
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