Localization of zein genes in maize

The band patterns of zein polypeptides were determined for many commercial inbred corn lines and maize stocks using isofocusing in agarose gels and sodium dodecyl sulfate (SDS)-urea gels. Each inbred line or homozygous maize strain genotype has a distinct zein profile which has been catalogued according to the distance of charge migration and molecular weight (kilodaltons). Several zein polypeptides were mapped to chromosomes 4 and 10 with the use of reciprocal translocations. The mapping of at least two polypeptides on distal 4L and 10L had not been previously reported. The general methods used in the present research will permit the mapping of all the zein polypeptides to chromosomal sites.Pioneer Hi-Bred International provided financial support.     

Complex organization of zein genes in maize

We have examined the fragments of maize nuclear DNA that are homologous to three cloned cDNAs prepared from zein mRNA. Southern blots of high molecular weight ( > 40 kb) maize nuclear DNA cleaved with BamHI, HindIII or EcoRI were hybridized to three zein cDNA plasmid probes. Multiple restriction fragments in a wide range of size classes were observed to hybridize with all three probes. Our results indicate the occurrence of families of genes in the maize genome that are related by their sequences to a given zein mRNA sequence.     

Adenine methylation of Okazaki fragments in Escherichia coli.

In Escherichia coli polA lig-4 bacteria, the moles percent 6-methyladenine content of 10S deoxyribonucleic acid (Okazaki fragments) is 0.96 compared with 1.4 for bulk desoxyribonucleic acid.     

Synthesis and cloning of artificial zein genes

To investigate structure-function relationships in zein proteins and to develop an approach to the construction of mutant zeins of improved nutritional qualities, the chemical-enzymatic synthesis of a gene for zein cZ22B1 (22 kd) has been undertaken. This 806-base pair long DNA fragment consists of about 40 synthetic oligonucleotides, mostly 30-60-mers. The synthesis was planned with the use of a universal methodology for the artificial gene construction. The choice of appropriate sites for altering amino acid sequence and the possibility of obtaining by directed mutagenesis of the gene corresponding to modified zeins containing residues Trp and Lys in specified positions of the polypeptide chain is discussed.     

Linkages among zein genes determined by isoelectric focusing

Summary Genetic control of the major zein polypeptides in maize (Zea mays L.) was studied by isoelectric focusing (IEF) in agarose. Linkage relationships were determined by making a number of crosses, then determining the expression of zein polypeptides in backcross seeds. Chromosome linkages were determined by using the markers sugary-1 (for chromosome 4), yellow-8, and a waxy 7–9 translocation (for chromosome 7). Nine zeins were in one linkage group on chromosome 4, six in another linkage group on chromosome 4, and four zeins were in one linkage group on chromosome 7. Some IEF single bands consisted of at least two polypeptides, which were detected by subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis, by aberrant ratios in backcrosses, or by differing recombination percentages. One zein occurred only in homozygous sugary-1 seeds. Three sets of closely-linked zeins were noted that occurred together almost exclusively in certain inbreds.Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the Illinois Agricultural Experiment Station, Department of Agronomy, University of Illinois, Urbana, USAMention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned     

Structure and expression of zein genes of maize

Plant seed storage proteins are synthesized and deposited in endosperm or cotyledon tissue to serve an important physiological function at the onset of germination. Because of their abundance, they constitute an important factor for the amount and nutritional value of kernel proteins. The physiological, biochemical, and genetic properties of many storage proteins and their genes, in particular those of cereals and legumes, have been extensively studied in the past and the results have been summarized in several reviews.^1–6 More recently, representative genes coding for storage proteins have been isolated and are now being used in attempts to elucidate the mechanism of the regulated synthesis of storage proteins. The purpose of this review is to outline, using maize as an example, the recent progress made in this effort.     

Adenine methylation may contribute to endosymbiont selection in a clonal aphid population

Background

The pea aphid Acyrthosiphon pisum has two modes of reproduction: parthenogenetic during the spring and summer and sexual in autumn. This ability to alternate between reproductive modes and the emergence of clonal populations under favorable conditions make this organism an interesting model for genetic and epigenetic studies. The pea aphid hosts different types of endosymbiotic bacteria within bacteriocytes which help the aphids survive and adapt to new environmental conditions and habitats. The obligate endosymbiont Buchnera aphidicola has a drastically reduced and stable genome, whereas facultative endosymbionts such as Regiella insecticola have large and dynamic genomes due to phages, mobile elements and high levels of genetic recombination. In previous work, selection toward cold adaptation resulted in the appearance of parthenogenetic A. pisum individuals characterized by heavier weights and remarkable green pigmentation.

Results

Six adenine-methylated DNA fragments were isolated from genomic DNA (gDNA) extracted from the cold-induced green variant of A. pisum using deoxyadenosine methylase (Dam) by digesting the gDNA with the restriction enzymes DpnI and DpnII, which recognize the methylated and unmethylated GATC sites, respectively. The six resultant fragments did not match any sequence in the A. pisum or Buchnera genomes, implying that they came from facultative endosymbionts. The A1 fragment encoding a putative transposase and the A6 fragment encoding a putative helicase were selected for further comparison between the two A. pisum variants (green and orange) based on Dam analysis followed by PCR amplification. An association between adenine methylation and the two A. pisum variants was demonstrated by higher adenine methylation levels on both genes in the green variant as compared to the orange one.

Conclusion

Temperature selection may affect the secondary endosymbiont and the sensitive Dam involved in the survival and adaptation of aphids to cold temperatures. There is a high degree of adenine methylation at the GATC sites of the endosymbiont genes at 8°C, an effect that disappears at 22°C. We suggest that endosymbionts can be modified or selected to increase host fitness under unfavorable climatic conditions, and that the phenotype of the newly adapted aphids can be inherited.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-999) contains supplementary material, which is available to authorized users.     

Analysis of distal flanking regions of maize 19-kDa zein genes

Two genomic fragments from maize, each containing a 19-kDa zein gene with extensive flanking regions, have been sequenced and examined by computer-aided analysis and Southern blotting techniques. Sequence analysis of the distal flanking sequences has revealed interesting sequence motifs, some not seen before. In particular, four nearly identical, G + C-rich, 17 to 21-bp perfect palindromes were found clustered in a 133-bp stretch lying 2 kb upstream from the zein-coding region in the genomic clone pMS2. These palindromic sequences exhibit other interesting features, including a precise spatial organization with respect to each other, and their proximity to several other repeated motifs in the same region. Southern blot analysis indicates that these palindromes, or closely related sequences, are found frequently in the maize genome. Possible secondary structures for the palindrome units are presented, which resemble functionally important sequences found upstream from other eukaryotic genes.     

Cloning and sequence analysis reveal structural variation among related zein genes in maize

We have isolated a gene encoding one of the 19,000 dalton zein proteins from a maize genomic library constructed in Charon 4A. This gene occurs on a 7.7 kb Eco RI fragment, and based on Southern hybridization analysis, represents one of several homologous sequences present in the maize genome. The nucleotide sequence of the gene predicts a protein composed of 235 amino acids, including a signal peptide of 21 amino acids. There are no intervening sequences in the gene. By comparing the nucleotide sequence of this gene with that of a homologous cDNA clone, we have identified a basis for microheterogeneity within the gene family. The 5′ nucleotide sequences of the genomic and cDNA clones are identical, but they differ in the center of the protein, where repeated amino acid sequences occur. A nucleotide sequence encoding a conserved peptide of 20 amino acids is repeated nine times in the center of both of these clones.     

Chromosomal localization of zein genes by in situ hybridization in Zea mays

Summary In order to localize the genes coding for zein, the major storage protein of maize endosperm, zein ¹²⁵I-mRNA and ³H-cDNA labelled at high specific activity were used for in situ hybridization on heterozygous interchanges and paracentric inversions of the KYS strain of Zea mays. The analysis of the diplotene-metaphase I microsporocytes indicated the presence of zein structural genes on the long arm of chromosomes 4 and 5, the short arm of chromosome 7 and the distal segment of the long arm of chromosome 10. The two hybridization sites on chromosomes 7 and 10 are found near opaque-2 and opaque-7 loci which are known to regulate zein synthesis. The present data are discussed in relation to results obtained by other authors using genetical mapping of zein genes.     

The manipulation of zein genes to improve the nutritional value of corn

The zein genes from corn (maize) were among the first plant genes to be isolated by recombinant DNA techniques. They encode a set of proteins which are of agricultural importance. Over 2 billion bushels of corn are fed to poultry and pigs each year in the USA alone. However the nutritional balance of corn is not ideal and thus farmers must buy expensive feed supplements. By changing the composition of zein proteins, genetic engineers might produce savings of many million dollars a year.