Linkage relationships between regulatory and structural gene loci involved in zein synthesis in maize

Summary The synthesis of at least 15 zein polypeptides is under the control of regulatory gene loci. One of these, Opaque-2 (chromosome 7, position 16) strongly reduces the zein accumulation without modifying the zein molecular components. The linkage relationship between the regulatory gene 02 and the 5 structural loci (Zp1, Zp2, Zp3, Zp6, Zp12) segregating with sample Mendelian ratios have been studied. Zp1, Zp2, Zp3 are closely linked to each other; moreover this gene cluster is located on chromosome 7 at 5.5 cM from the Opaque-2 locus. The structural loci Zp6 and Zp12 are not linked with each other, with the 02 locus or with Zp1, Zp2, Zp3. From our data it follows that the zein structural genes are located in at least three positions on the maize genome. The scattering in the genome of the genes controlled by the Opaque-2 locus suggests a transacting role for this regulatory element.     

Clustering of genes for 20 kd zein subunits in the short arm of maize chromosome 7

Zein is the major storage protein of the endosperm of maize kernels. When this alcohol-soluble protein is subjected to SDS polyacrylamide gel electrophoresis, it is resolved into four fractions of different molecular weight: 10, 14, 20 and 22 kilodaltons (kd). Each fraction is heterogeneous with respect to isoelectric pH. For example, the 20 kd fraction contains at least seven subfractions as revealed by isoelectric focusing in polyacrylamide gels. In this report, we present evidence that the structural genes coding for the 20 kd proteins are clustered on the short arm of chromosome 7, a region that also bears loci regulating endosperm zein biosynthesis [opaque-2 (02) and defective endosperm-B30 (De^*-B30)]. The organization of these zein genes suggests that the evolution of at least some of the maize genome has occurred as the result of repeated duplication and divergence of chromosome segments.     

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.     

Comparison between responses to gametophytic and sporophytic recurrent selection in maize (Zea mays L.)

Summary Experiments were conducted to determine the chromosomal location of the gene conditioning overproduction of a methionine-rich, 10-K zein in maize kernels of line BSSS53. In addition, the chromosomal location of the structural gene encoding the overproduced protein was determined. Whereas the structural gene, designated Zps10/(22), was found to be located on the long arm of chromosome 9 near the centromere, the locus regulating overproduction of the zein protein was mapped to the short arm of chromosome 4. This regulatory gene has been designated Zpr10/(22). Regulation of 10-K zein production by Zpr10/(22) is, therefore, via a trans-acting mechanism.     

Effect of sucrose accumulation on zein synthesis in maize starch-deficient mutants

Starch-deficient maize (Zea mays) mutants, brittle-2 (bt2), brittle-1 (bt), and shrunken-2 (sh2), which accumulated large quantities of sucrose, had less than normal amounts of zein (the major storage protein) in the endosperm. Reduction of zein synthesis in the starch-deficient mutants was negatively correlated with the accumulation of sucrose and low osmotic potential in the developing endosperms. When radioactive amino acids were injected into the shank below ears that segregated for the starch-deficient mutant and normal kernels at 28 days post-pollination, mutant kernels absorbed only ca 22–36% of the labelled amino acids found in their normal controls. Thus, a low osmotic potential in the mutant endosperm may favour water movement but reduce solute movement. The inability of amino acids to move into the mutant endosperms, therefore, in part explains the reduction of zein accumulation in starch-deficient mutant endosperms.     

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.     

Synthesis and deposition of zein in protein bodies of maize endosperm

The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [¹⁴C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.

Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained.

     

Genetic basis of the major malate dehydrogenase isozymes in maize

The mitochondrial MDH isozymes in the scutellum of the mature maize (Zea mays L.) kernel are encoded by three independently inherited nuclear genes. Mdh1 is located on chromosome 8, close to the breakpoint (8L.35) of a waxy-marked reciprocal translocation between chromosomes 8 and 9. Mdh2 is located in the distal region of the long arm of chromosome 6. Mdh3 is on the long arm of chromosome 3, approximately 2.6 map units from sh2. A modifier of the mitochondrial MDH isozymes (Mmm) maps approximately 27.5 units proximal to Adh1 in the central portion of the long arm of chromosome 1. Independently assorting duplicate genes code for the soluble MDH isozymes. Mdh4 is located in the same region of chromosome 1 as Mmm, approximately 29 map units proximal to Adh1. Mdh5 maps approximately 20 units distal to a2 in the short arm of chromosome 5.——Intergenic and interallelic heterodimer formation occurs among gene products that occupy the same subcellular compartment. MDH isozymes were purified and analyzed by native-SDS two-dimensional polyacrylamide gel electrophoresis. The proposed mitochondrial MDH intergenic heterodimer bands were found to be composed of two subunits, which differ in their migrations on SDS gels; whereas, genetically defined homodimers contained only one type of subunit.——This evidence is discussed in terms of two genetic models proposed for the maize mitochondrial MDH isozymes.     

Serial analysis of zein by isoelectric focusing and sodium dodecyl sulfate gel electrophoresis

Zein, the major storage protein of maize (Zea mays L.) endosperm, was extracted from a number of inbreds with alcohol plus a reducing agent. Isoelectric focusing (IEF) separated total zeins into 41 components, while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separated total zeins into about 15 components. Each procedure gave characteristic patterns of zein bands for a number of maize inbreds. IEF and SDS-PAGE were used serially so that each band separated by IEF could be assayed as an individual SDS-PAGE sample. Some IEF bands revealed only a single band after SDS-PAGE, while others revealed two or more bands. A nomenclature system is presented which integrates the two separation systems with information about chromosome locations of zein genes, maize mutations which affect zein synthesis, and inbred sources for different zeins. SDS-PAGE of zein gives apparent molecular masses which vary widely according to the standards used and the properties of the gels, therefore an artificial nomenclature for identifying zein bands after SDS-PAGE is presented. The new nomenclature provides a flexible system which is useful and can be conveniently used in different laboratories.     

Contiguous genomic DNA sequence comprising the 19-kD zein gene family from maize

A new approach has been undertaken to analyze the sequences and linear organization of the 19-kD zein genes in maize (Zea mays). A high-coverage, large-insert genomic library of the inbred line B73 based on bacterial artificial chromosomes was used to isolate a redundant set of clones containing members of the 19-kD zein gene family, which previously had been estimated to consist of 50 members. The redundant set of clones was used to create bins of overlapping clones that represented five distinct genomic regions. Representative clones containing the entire set of 19-kD zein genes were chosen from each region and sequenced. Seven bacterial artificial chromosome clones yielded 1,160 kb of genomic DNA. Three of them formed a contiguous sequence of 478 kb, the longest contiguous sequenced region of the maize genome. Altogether, these DNA sequences provide the linear organization of 25 19-kD zein genes, one-half the number previously estimated. It is suggested that the difference is because of haplotypes exhibiting different degrees of gene amplification in the zein multigene family. About one-half the genes present in B73 appear to be expressed. Because some active genes have only been duplicated recently, they are so conserved in their sequence that previous cDNA sequence analysis resulted in "unigenes" that were actually derived from different gene copies. This analysis also shows that the 22- and 19-kD zein gene families shared a common ancestor. Although both ancestral genes had the same incremental gene amplification, the 19-kD zein branch exhibited a greater degree of far-distance gene translocations than the 22-kD zein gene family.     

Computational Finishing of Large Sequence Contigs Reveals Interspersed Nested Repeats and Gene Islands in the rf1-Associated Region of Maize

The architecture of grass genomes varies on multiple levels. Large long terminal repeat retrotransposon clusters occupy significant portions of the intergenic regions, and islands of protein-encoding genes are interspersed among the repeat clusters. Hence, advanced assembly techniques are required to obtain completely finished genomes as well as to investigate gene and transposable element distributions. To characterize the organization and distribution of repeat clusters and gene islands across large grass genomes, we present 961- and 594-kb contiguous sequence contigs associated with the rf1 (for restorer of fertility1) locus in the near-centromeric region of maize (Zea mays) chromosome 3. We present two methods for computational finishing of highly repetitive bacterial artificial chromosome clones that have proved successful to close all sequence gaps caused by transposable element insertions. Sixteen repeat clusters were observed, ranging in length from 23 to 155 kb. These repeat clusters are almost exclusively long terminal repeat retrotransposons, of which the paleontology of insertion varies throughout the cluster. Gene islands contain from one to four predicted genes, resulting in a gene density of one gene per 16 kb in gene islands and one gene per 111 kb over the entire sequenced region. The two sequence contigs, when compared with the rice (Oryza sativa) and sorghum (Sorghum bicolor) genomes, retain gene colinearity of 50% and 71%, respectively, and 70% and 100%, respectively, for high-confidence gene models. Collinear genes on single gene islands show that while most expansion of the maize genome has occurred in the repeat clusters, gene islands are not immune and have experienced growth in both intragene and intergene locations.Genome sequencing of the maize (Zea mays) genome is nearing completion (Bennetzen et al., 2001; Chandler and Brendel, 2002; Wessler, 2006); it is the largest and most difficult-to-assemble plant genome sequenced to date. Maize is an important economic, agricultural, industrial, and research crop; however, with a genome close to the size of the human genome (2.8 Gb) and its high percentage of repetitive elements, acquiring the maize genome seemed a daunting task. Approximately 67% of the genome is made up of transposable elements (TEs; Haberer et al., 2005; Kronmiller and Wise, 2008), increasing the difficulty of assembly (Rabinowicz and Bennetzen, 2006). Much exploratory work has gone into isolating and sequencing just the gene areas and ignoring the repetitive regions, both by methylation filtration (Rabinowicz et al., 1999; Palmer et al., 2003; Whitelaw et al., 2003) and high-C₀t (Whitelaw et al., 2003; Yuan et al., 2003) systems, which have assisted researchers with selecting only genic regions to sequence. These methods have captured a majority of the maize genic sequence (Fu et al., 2005), but they still have the potential to miss important regions. The current genome-sequencing project aims to capture the entire gene set of maize, including regulatory regions. However, the current strategy will not provide a fully assembled genome but rather assembled bacterial artificial chromosome (BAC) contigs ordered and orientated to provide complete gene regions that are adjacent to potentially incomplete TE clusters.The landscape of the maize genome provides an interesting challenge for both sequencing and subsequent annotation. A high density of long terminal repeat (LTR) retrotransposons has had a direct effect on the genome size of many plant genomes, including maize (SanMiguel et al., 1996; Bennetzen et al., 2005; Hawkins et al., 2006; Piegu et al., 2006). Besides expanding genome size, LTR retrotransposons can have an impact on evolution of the species (Kidwell and Lisch, 2000). LTR retrotransposon insertions tend to form nested clusters (SanMiguel and Bennetzen, 1998), which are separated by small regions of several genes. Large nested repeat clusters consist of TE insertions inside TE sequences, expanding the repeat cluster and breaking up the sequence of the TEs found within, hindering repeat and gene annotation and increasing the difficulty of assembly. However, full sequence completion of the repetitive regions can be of great benefit to understanding the evolutionary history of the maize genome. LTR retrotransposons can provide an estimated time since insertion by calculating the divergence of their LTRs (Kimura, 1980; Ma and Bennetzen, 2004), and carefully sequenced assemblies of nested repeat clusters can help to illustrate their expansion, proliferation, and evolution across the genome (Kronmiller and Wise, 2008).Previous studies of large contiguous regions of maize have provided a general view of the landscape of the genome. Unfinished sequence totaling 7.8 Mb from chromosome 1 and 6.6 Mb from chromosome 9 shows a gene density of one gene per 33 and 27 kb, respectively (Bruggmann et al., 2006). BAC contigs ranging in size from 126 to 405 kb show a gene density of one gene per 19 kb and genes found in small groups between large repeat clusters (Brunner et al., 2005). Genome-wide analysis of maize BACs has painted a different picture: while gene density of 100 random BACs at one gene per 44 kb was similar to the above results, genes were not observed in tight clusters (Haberer et al., 2005). When investigating gene-specific areas of maize, this dichotomy of gene density is also seen. Analysis of gene-rich regions such as the 22-kd α-zein gene family on maize chromosome 4 reveals a high density of genes, with one gene observed per 10 kb over 346 kb (Song et al., 2001). The Adh1 locus on maize chromosome 1 contains two genes across 280 kb, or one gene per 140 kb. Perhaps the only message learned here is that the gene density across the maize genome varies to a great degree, and large contiguous sequenced regions can begin to capture the true diversity of maize chromosome architecture.In order to characterize large contiguous regions of maize sequence, we identified and sequenced two B73 BAC contigs from the centromeric region of chromosome 3. These contigs of 961 and 594 kb correspond to contigs 117 and 119, respectively, on maize WebFPC (Wei et al., 2007) and span regions associated with the rf1 (for restorer of fertility1) locus for Texas (T) cytoplasmic male sterility (cmsT; Duvick et al., 1961; Wise et al., 1996). As a foundation for the isolation of the Rf1 locus, four rf1 male-sterile mutants were recovered from a screen of 123,500 flowering plants (Wise et al., 1996). A 5.5-kb Mu1-hybridizing EcoRI restriction fragment was identified that cosegregated with the rf1-m3207 allele. Sequences from this fragment were hybridized to a Rf1 cDNA library, and probes designed from the identified cDNA, p6140-1 (Wise et al., 1999), were found to cosegregate with the rf1 locus in a recombinant population selected from over 10,000 progeny.Using probes designed off the 5.5-kb cosegregating restriction fragment and the p6140-1 cDNA, we have identified two BAC contigs spanning the rf1 locus. Sixteen BACs were sequenced to completion to provide high-quality finished sequence. Here, we present two methods for computational finishing of highly repetitive grass genomes, which were successfully utilized to close 11 TE-induced gaps. Sixteen nested repeat clusters were found, each spanning as much as 155 kb and containing a variety of LTR retrotransposon types and ages of insertion. Genes are found tightly clustered, showing a density rate of one gene per 16 kb within gene islands. Finally, comparative analysis with rice (Oryza sativa) and sorghum (Sorghum bicolor) shows that while many genes are retained across all three species, genes have both been lost and translocated across the genomes.     

Characterization of Polypeptides Corresponding to Clones of Maize Zein mRNAS

Zeins, the storage proteins of maize (Zea mays) are a complex group of polypeptides encoded by a large multigene family. The α-zein proteins, which account for about 70% of the total, show both size and charge heterogeneity. Although clones corresponding to several different alpha zeins have been characterized, it has not been possible to correlate these sequences with individual zein polypeptides. By translating in Xenopus oocytes RNAs transcribed in vitro from cloned zein mRNAs, we were able to identify the encoded proteins among native zeins or zeins synthesized in oocytes with total zein mRNA. There was no correlation between the isoelectric points of these proteins and the homology of their coding DNA sequences, as the proteins encoded by two closely homologous cDNAs migrated with greater charge heterogeneity than those encoded by less homologous clones. In addition, the size of the proteins as determined by SDS polyacrylamide gel electrophoresis did not always correlate with the length of the protein deduced from the DNA sequence. The ability to match cloned zein sequences to individual native proteins will enable the genetic mapping of cloned genes as well as the analysis of their translational regulation.