Distribution of sequences related to the Bg transposable element of maize in Zea and related genera.

Thirty-four accessions from Zea and 10 accessions from related genera were assayed for the presence of Bg, a transposable element originally found in maize (Zea mays ssp. mays). Bg-like sequences, identified as hybridizing bands on Southern blots, were visualized in all Zea accessions and were present in approximately equal numbers in teosinte and maize. With the exception of Tripsacum dactyloides, all accessions from related genera failed to hybridize with the Bg probes, even at reduced stringency. A comparison of the restriction patterns of related inbred lines revealed numerous common hybridizing fragments. An index of molecular similarity (MS) was used to determine the degree of similarity between pairs of inbred lines. Computed MS values endorse an inbred relationship and are in good agreement with published results of cluster analysis on these inbred lines.

Molecular analysis of opaque-2 alleles from Zea mays L. reveals the nature of mutational events and the presence of a hypervariable region in the 5' part of the gene.

Ten recessive Opaque-2 (O2) alleles of independent origin were characterized at the molecular level. The results revealed a high level of polymorphism at the O2 locus. In addition, our data suggest the possible cause for the recessive character of some of the alleles investigated, and allow us to infer some conclusions concerning the degree of relationship between the o2 mutations. Comparison of genomic sequences spanning the first exon and obtained from a series of wild-type and recessive alleles revealed the presence of a hypervariable region, involving different dipeptides, in the N-terminal part of the O2 protein.

Structural and functional analysis of an Opaque-2-related gene from sorghum.

The Opaque-2 (O2) gene from maize encodes a transcriptional activator of the b-ZIP class. We have isolated and characterized a gene from sorghum, related in sequence to the O2 gene from maize. A single copy of the gene is present in sorghum. Both genomic and cDNA sequences of the O2-related sorghum gene were determined. The sequence is highly homologous to maize O2 both in the promoter and in the coding region. The most closely related sequences contain the b-ZIP domain with only 11 amino acid substitutions in a total of 122 residues. In transient expression assays, the sorghum O2-related coding sequence, expressed from a CaMV 35S promoter, activates expression from the maize b-32 promoter as effectively as that obtained with the maize O2 sequence.

Molecular analysis of the Bg-rbg transposable element system of Zea mays L.

The two components of the Bg-rbg transposable element system of maize have been cloned. The Bg element, isolated from the mutable allele wx-m32:: Bg is inserted in the intron of the Waxy (Wx) gene between exons 12 and 13. The length of the element is of 4869 bp. Bg has 5 bp terminal inverted repeats, and generates upon insertion an 8 bp direct duplication of the target sequence. Both ends of the Bg element contain a 76 bp direct repeat adjacent to the terminal inverted repeats. The hexamer motif TATCGGC is here repeated several times in direct or inverse orientation. The rbg element was isolated from the mutable allele o2m(r) where it is located in the promoter region of the Opaque-2 (O2) gene. rbg is approximately 4.5 kb in length, has terminal inverted repeats identical to those of the Bg element, and is also flanked by an 8 bp direct duplication at the target site. Like Bg, rbg carries the 76 bp direct repeats. Restriction enzyme analysis reveals that, compared to Bg, the receptor element is distinguishable by small deletion and insertion events. Sequence data indicate that not more than 75% homology exists at the DNA level between the rbg element and the autonomous Bg element.

The b-32 protein from maize endosperm: characterization of genomic sequences encoding two alternative central domains.

As derived from a cDNA clone, the structure of the b-32 protein of Zea mays, a putative regulatory factor of zein expression, has a central acidic region separated by two domains covered by secondary structure motifs. In this work, three b-32 genomic clones were selected from two genomic libraries obtained from the maize inbred lines W64A and A69Y. The nucleotide sequences of the complete coding region of each b-32 gene, as well as long stretches of their 5' and 3' flanking regions, were determined. Introns are not present in the b-32 genomic sequences. Minor variations among the three genes and an earlier reported b-32 cDNA indicates that they constitute a gene family showing a characteristic polymorphism. Such a polymorphism is highly evident in large segments of the upstream regulatory sequences. Interestingly, when compared with cDNA (W64A) or with gene b-32.120 (W64A), the genes b-32.129 (W64A) and b-32.152 (A69Y) show three jumps of the reading frame in the central part of the coding region, resulting in a completely different sequence of the b-32 protein central domain. In all cases, variations in the N- and C-terminal domains account only for microheterogeneity.

The O2 gene which regulates zein deposition in maize endosperm encodes a protein with structural homologies to transcriptional activators.

The structure of the zein regulatory gene Opaque 2 of Zea mays has been determined by sequence analysis of genomic and cDNA clones. The size of O2 mRNA is 1751 bp [poly(A) tail not included] containing a major open reading frame (ORF) of 1380 bp preceded by three short ORFs of 3, 21 and 20 amino acid residues. The main ORF comprises 1362 bp and is composed of six exons ranging in size from 465 to 61 bp and five introns of 678 bp to 83 bp. A putative protein 454 amino acids long was derived by the theoretical translation of the genomic sequences corresponding to exons. The opaque 2 protein contains a domain similar to the leucine zipper motif identified in DNA binding proteins of animal protooncogenes such as fos, jun and myc, and in the transcriptional activators GCN4 and C/EBP. The region of 30 amino acid residues next to the leucine repeats towards the N terminus is rich in basic amino acids and is also homologous to a domain present in fos, jun and GCN4. Moreover, in the carboxy terminal region an amino acid motif closely resembling a metal binding domain is present.