Mapping of theHor2 locus in barley by pulsed field gel electrophoresis

Sørensen, Mikael Blom

doi:10.1007/bf02908303

Cited by 23 publications

(4 citation statements)

References 44 publications

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“…DNA was labeled by random priming (17) and separated from unincorporated nucleotides by using a Sephadex G-50 column (Nick-columns, Pharmacia). Prehybridization, hybridization, and washing of filters were as described (18 …”

Section: Methodsmentioning

confidence: 99%

Methylation of B-hordein genes in barley endosperm is inversely correlated with gene activity and affected by the regulatory gene Lys3.

Sørensen

1992

Proc. Natl. Acad. Sci. U.S.A.

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The methylation status of B-hordein genes in the developing barley endosperm was analyzed by digestion with methylation-sensitive restriction enzymes. Southern blotting revealed specific demethylation of Hpa II sites in DNA from wild-type endosperm, whereas leafDNA and lys3a mutant endosperm DNA were highly methylated at these sites. Similar methylation patterns were observed at an Ava I site situated at position -260 in the B-hordein promoter. This differential methylation was confirmed by genomic sequencing with ligation-mediated PCR. The analyzed sequence covers most of the B-hordein promoter and includes 10 CpGs from the promoter and 4 CpGs from the adjacent coding region. These sites were all hypomethylated in wild-type endosperm, whereas-except for three partially methylated sites-full methylation was seen in leaf DNA. The four sites in the coding region were partially methylated in lys3a endosperm DNA, but the promoter sites remained highly methylated. The possible role of methylation in the regulatory function of the Lys3 gene product is discussed.

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Section: Methodsmentioning

confidence: 99%

Methylation of B-hordein genes in barley endosperm is inversely correlated with gene activity and affected by the regulatory gene Lys3.

Sørensen

1992

Proc. Natl. Acad. Sci. U.S.A.

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“…Of these the Hor2 locus on chromosome 5, the gene family encoding B-hordein polypeptides, has been studied in detail. The locus comprises about 13 structural genes covering at least 360 kbp [41]. Since the similarity at the nucleotide level of the Pazl-related fragments is low, we are reluctant to consider the additional fragments as members of a Paz multigene family.…”

Section: Discusslonmentioning

confidence: 99%

A plant serpin gene

Brandt¹,

Svendsen

Hejgaard

1990

European Journal of Biochemistry

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A 3133-bp nucleotide sequence of the gene Pazl on chromosome 4 of barley, encoding endosperm protein Z4, has been determined. The sequence includes 1079 bp 5' upstream and 523 bp 3' downstream of the coding region. The 1079-bp 5' upstream region of the gene shows little similarity to 5' regions of other sequenced genes expressed in the developing cereal endosperm. The coding sequence is interrupted by one 334-bp-long intron (bases 1497 -1830). The deduced amino acid sequence, which was corroborated by peptide sequences, consists of 399 amino acids and has a molecular mass of 43 128 Da. This sequence confirms protein Z4 to be a member of the serpin superfamily of proteins. The similarity with other members of the family expressed as amino acids in identical positions is in the order of 25-30% and pronounced in the carboxy-terminal half of the molecule. Sequence residues assumed to form clusters stabilizing the tertiary structure are highly conserved. Protein Z4 is synthesized in the developing endosperm without a signal peptide and protein Z4 mRNA was evenly distributed among the free and membrane-bound polyribosomes of the endosperm cell. An internal hydrophobic region of 21 amino acids (residues 36 -56) may serve as a signal for targeting the polypeptide into the lumen of the endoplasmic reticulum. The gene for protein Z4 could not be detected in the barley variety Maskin and some of its descendants.The 'high-lysine' allees, lysl (Hiproly barley) and lys3a (Bomi mutant 1508) on chromosome 7, enhance and repress, respectively, the expression of the protein Z4 gene. Also, 1554 bp of another 8-kbp fragment of the barley genome Paz Y , similar to the protein-Z4-coding region, have been determined. Small insertions and deletions and the presence of an internal stop codon identify this fragment as part of a pseudogene related to the protein Z4 gene.Barley protein Z4 is an x 43-kDa polypeptide of the endosperm, accounting for about 5% of the albumin fraction [l]. The polypeptide is synthesized in the developing endosperm 10 -25 days after fertilization, together with the major storage proteins, the hordeins [2, 31. Like the hordeins, protein Z4 synthesis is enhanced when increasing amounts of nitrogen fertilizer are supplied to the barley plant during development of the caryopsis [4, 51. Protein Z4 is accumulated and stored in the endosperm, but the site of synthesis and deposition within the cell is not known. The polypeptide is resistant to proteases synthesized and secreted by the aleurone and scutellum tissue during early stages of germination [6]; originally, protein Z4 was isolated as an M 40-kDa polypeptide from beer [7].Barley protein Z4 is coded for by a locus on chromosome 4, designated Pazl [8]. A less abundant, immunologically related polypeptide, protein Z7, is coded for by a locus on chromosome 7 [9]. Two 'high-lysine' alleles, lysl and lys3a on chromosome 7, increase and decrease the synthesis of protein Correspondence to I. Svendsen, Department of Chemistry, Carlsberg Laboratory, Gamle Carlsbergvej 10,...

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“…This was verified by Anders Brandt, John Ingversen, Verena Cameron-Mills, and Susan Madrid using isolated endoplasmatic reticulum (5,6,(11)(12)(13) and isolated protein bodies and by localization studies with monoclonal antibodies developed by Gunilla Høyer-Hansen (58,65,66,77) (see Supplemental Figures 2 and 3). Cloning and sequencing of structural genes for B-, C-, and γ-hordein polypeptides have corroborated that the Hor 1, Hor 2, and Hor F loci each consist of a family of closely linked structural genes for these proteins, with each gene having its own promoter (7,10,18,24,62,63,70). Some of these genes contain stop codons, but transient transformation experiments revealed that expression of such a gene in the endosperm can take place by amber codon suppression (19).…”

Section: Prefacementioning

confidence: 99%

From Analysis of Mutants to Genetic Engineering

Wettstein

2007

Annu. Rev. Plant Biol.

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This chapter describes the research of developing transgenic barley for synthesis of recombinant proteins with practical significance and of metabolic engineering of proanthocyanidin-free barley. The results were obtained by graduate students, postdoctoral researchers, and visiting scientists at the Carlsberg Laboratory from 1972-1996 and during the past ten years at Washington State University. It is written in appreciation of their enthusiasm, skill, and perseverance.

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Mapping of theHor2 locus in barley by pulsed field gel electrophoresis

Cited by 23 publications

References 44 publications

Methylation of B-hordein genes in barley endosperm is inversely correlated with gene activity and affected by the regulatory gene Lys3.

Methylation of B-hordein genes in barley endosperm is inversely correlated with gene activity and affected by the regulatory gene Lys3.

A plant serpin gene

From Analysis of Mutants to Genetic Engineering

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