Binding of a nuclear factor to a consensus sequence in the 5' flanking region of zein genes from maize

The genomic organization of the zein structural genes and of regulatory loci influencing their expression suggests that control of zein gene expression will involve interactions between cis elements in the flanking DNA sequences and products from trans-acting genes. The interaction between fragments from the 5' flanking region of a zein gene and specific, double-stranded oligonucleotides with crude nuclear extracts from maize endosperm have been studied by nitrocellulose filter binding, gel retention and DNase I footprinting assays. Specific binding of a nuclear factor was observed and the exact position of the protein binding site was determined. The 22-nt binding site included 14 bp of a 15-bp sequence conserved in all zein genes.     

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.     

Zein gene organization in maize and related grasses

Summary Zein cDNA clones were used to study the organization of zein genes within the genome of the inbred maize W64A. When individual clones for the two larger molecular-weight classes of zein proteins (M_r=22,000; M_r=19,000) were used as probes for Southern blot hybridizations of genomic DNA, multiple restriction fragments were found to hybridize. Reconstruction analyses using moderately stringent criteria were used to estimate a total of 70–80 zein sequences within the genome of this inbred maize. The hybridization patterns suggest that zein sequences are clustered within the same restriction fragment. When criteria permitting less cross-hybridization of homologous sequences (T_m-10°C) were used, the banding pattern changed, with some of the bands being reduced in intensity or eliminated entirely. Therefore, by control of hybridization criteria, particular zein genes may be more readily distinguished in a Southern blot analysis. The Southern blot hybridization pattern for the M_r=15,000 zein was less complex. Only a single major band was found, with sufficient hybridization intensity for two or three genes.Genomic Southern analyses of other inbred maizes and related grasses showed similarly complex hybridization patterns with cDNA probes for the 19,000- and 22,000-molecular-weight zeins, suggesting that these sequences have been conserved over evolutionary time. The zein multigene family may therefore have arisen by gene duplication before divergence of the maize, teosinte, andTripsacum species from a common ancestor.This is Journal Paper number 9525 of the Purdue Agriculture Experiment Station     

Isolation and characterization of a genomic sequence of maize coding for a zein gene

Summary An EcoRI restriction endonuclease fragment of maize DNA coding for the 19,000 dalton zein protein was cloned in phage gt WES. The zein gene was identified by the electron microscopic analysis of RNA-DNA hybrids (R-loops) and DNA-DNA hybrids (D-loops). The R-loops were formed with poly(rA)-containing RNA isolated from 18 days post-pollination maize endosperm and showed no intervening non-hybridizing sequences (introns) within their 800 base length. A cDNA clone specific for the 19,000 dalton zein protein formed D-loops in the same position and orientation as the R-loops. The cloned fragment measured 4.4 kilobases (kb), the same size as an EcoRI fragment of maize DNA revealed by Southern analysis.     

Multiplicity and diversity of cloned zein cDNA sequences and their chromosomal localisation

We have constructed and screened cDNA libraries from total maize endosperm poly(A) RNA or from a mRNA fraction enriched in zein sequences. From these libraries we have isolated clones representative of the major classes of zein cDNA sequences and have characterised them by crosshybridisation, by hybrid-selected translation, by in situ hybridisation to maize chromosomes, and hybridisation to genomic Southern blots. We conclude that at least four types of non cross-hybridising zein sequences are present, two coding for light chains and two for heavy chains. At least in the case of the light zeins, there is considerable sequence diversity among the clones which hybridise to each type. Similar results are obtained by translation of the mRNAs selected by each clone. In situ hybridisation shows that the light chain zein genes are located on chromosomes 4, 7, and 10, whilst genes coding for some of the heavy chain zeins are confined to the distal part of the long arm of chromosome 4.     

Amplicons of maize zein genes are conserved within genic but expanded and constricted in intergenic regions

The 78 101 base pair long sequence of a cluster of 22-kDa alpha zein genes in the maize inbred BSSS53 was determined. Each zein gene is contained within a repeat unit that varies in length. If such a repeat, or amplicon, is aligned along the entire sequence, a 10.5-fold sequence amplification is delineated. Because of insertions and deletions in intergenic regions, many of the zein genes are spaced over different distances. Only three out of 10 zein-related sequences have an intact open reading frame, indicating an unusual large number of genes unable to contribute to the accumulation of normal-size 22-kDa zein proteins. It is proposed that the seven remaining zein-related sequences be considered gene reserves because of their potential to be restored by gene conversion. Intergenic insertions in the cluster range from 1098 to 14 896 base pairs. Although they are composed of transposable element sequences, they also contain additional open reading frames, two of them showing homology to rice cDNA sequences. The average amplicon is 4423 base pairs long, with the sequence surrounding each zein gene more than 90 % conserved. Coincidently, the size of the amplicon is equivalent to the average gene density (one gene within 4640 bp) in the Arabidopsis thaliana genome, one of the smallest in plants. Successive steps of amplification and insertion of DNA might explain to a certain degree how genome size variation has been generated in plants.     

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.     

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.     

The effects of deoxycholate and trypsin on the cross-linking of rabbit skeletal muscle sarcoplasmic reticulum proteins.

Dithiobis (succinimidyl propionate) has been used to cross-link sarcoplasmic reticulum microsome proteins. Although the 100,000 dalton calcium stimulated ATPase and the 60,000 dalton calcium-binding protein calsequestrin were readily cross-linked to form homopolymers, no heteropolymer formation between these two proteins were detected. The 90,000 dalton protein A1 which is always observed in our preparations appeared to preferrentially form dimers on cross-linking. When calsequestrin was solubilized using 0.1 mg deoxycholate/mg protein, this protein was not cross-linked even at dithiobis(succinimidyl propionate) concentrations ten times those used to cross-link this protein in the intact membrane. In a similar manner the deoxycholate-solubilized ATPase (0.5 mg deoxycholate/mg protein) was not cross-linked by dithiobis (succinimidyl propionate). These results suggest that the state of aggregation of the sarcoplasmic reticulum proteins may be modified when solubilized in detergents such as deoxycholate. When the 100,000 dalton ATPase polypeptide was cleaved with trypsin to two fragments with molecular weights of approximately 55,000, these could be readily cross-linked. The fragments were capable of forming polymers with either other 55,000 dalton fragments or with the 100,000 dalton ATPase. The 29,000 and 22,000 dalton fragments, produced by further tryptic cleavage of the 55,000 dalton fragments, were not cross-linked at dithiobis (succinimidyl propionate) concentrations which readily cross-linked the 55,000 dalton fragments. Thus tryptic cleavage of the ATPase to fragments smaller than 55,000 dalton altered associations made by the ATPase in the membrane.     

Sequence analysis and characterization of a maize gene encoding a high-sulfur zein protein of Mr 15,000

We have isolated a gene coding for a sulfur-rich zein protein from a maize genomic library. The nucleotide sequence of this gene predicts a protein composed of 180 amino acids, including a 20-amino acid signal peptide. As is true of other zeins, there are no intervening sequences in the gene. Comparison of the nucleotide sequence of this gene with that of a homologous cDNA clone revealed only a single difference resulting in a valine/alanine substitution. The Mr 15,000 zein contains no repetitive nucleotide sequences and shows no homology with genes encoding the Mr 22,000 and 19,000 zeins. Circular dichroism analysis of the Mr 15,000 zein protein revealed that it is composed primarily of beta and turn structures. This gene has a short region of nucleotide sequence homology to the cysteine-rich domain of the Mr 27,000 zein, as well as the cysteine-containing barley B-hordein.     

Two adjacent genomic zein sequences: structure, organization and tissue-specific restriction pattern

A clone isolated from a library of genomic clones from Zea mays contains two adjacent zein light-chain genes. The nucleotide sequence of the two coding regions and their flanking sequences shows that the two genes represent two different subclasses of light-chain zeins. One belongs to an abundant and highly conserved class and the other, which is 88% homologous to the first, is less represented in the genome of maize line W64A. Both genes present in clone zE are anomalous: one contains a mutation that introduces a termination codon after 39 amino acids, the other has the ATG initiation codon mutated to CTG. The presence of inversions, insertions and short inverted or direct repeats in the flanking sequences suggests that zein genes may be subject to rearrangements and transpositions. Southern blot analysis of genomic DNA indicates that the particular arrangement represented by clone zE is present at least four times in maize line W64A but other arrangements are found in this and other maize lines. DNA extracted from different tissues and developmental stages is digested differently by enzymes sensitive to methylation of their target sequence. The results indicate that zein genes contain methylated sites that are demethylated in correlation with their expression.     

Isolation and characterisation of the Xenopus laevis albumin genes: loss of 74K albumin gene sequences by library amplification.

The blood of the frog X.laevis contains 2 albumins of 68,000 and 74,000 daltons which are encoded in the liver by two related mRNAs. When an amplified X.laevis DNA library was screened with cloned albumin cDNA only 68,000 dalton albumin gene sequences were isolated. Hybridisation of the albumin cDNA to Southern-blots of Eco R1 digested X.laevis DNA showed that the sequences present in the recombinants did not account for all the fragments which hybridised on the Southern-blots. This indicated that 74K albumin gene sequences exist but that they are not present in the amplified DNA library. A X.laevis genomic library was therefore constructed and screened for albumin genes without amplification. Both 68K and 74K albumin gene sequences were isolated. Recombinants containing 74K albumin gene sequences grew extremely poorly and this probably explains why the 74K albumin sequences were ot isolated from the amplified library. Characterisation of the cloned DNA indicates that there is one sequence coding for the 68K albumin but two different sequences coding for the 75K albumin.     

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.