Günter Feix

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.

Early post-embryonic root formation is specifically affected in the maize mutant lrt1.

The isolation and detailed characterisation of the maize mutant lrt1 , which is completely deficient in the initiation of lateral roots at the primary and seminal lateral roots and of the crown roots at the coleoptilar node is described. The monogenic and recessive mutant was isolated from a segregating EMS mutagenised population, maps to the short arm of chromosome 2, and acts independently of the nodal root deficient rtcs locus. Histological analysis revealed that the mutation acts at a very early stage of root initiation, as indicated by the absence of primordia formation in the affected roots. At later stages of plant development lateral and crown root initiations recover leading to fertile plants. If grown in the dark, the mutant does not form an elongated mesocotyl, although the photomorphogenic response appears to be normal in the mutant. Furthermore, the wild-type cannot be rescued from mutants by the application of auxin to germinating kernels. The gene impaired in lrt1 seems to be of great importance for the general mechanism of early post-embryonic root initiation, both from root and nodal tissues, since lateral and crown root initiation are both affected to the same extent and in the same transient time pattern.

A zein gene of maize is transcribed from two widely separated promoter regions

The zein proteins are the major storage proteins of maize endosperm. They are made from large amounts of zein RNAs of several distinct sizes, ranging from the small 900 base final size RNA to precursor forms of over 3800 bases. The 900 base mRNA and the smallest precursor mRNA of 1800 bases are transcribed from two distinct promoter regions, P1 and P2. In vitro transcription of a maize genomic clone containing a zein gene pML 1 allowed both promoters to be mapped. The sequence of pML 1, covering the two promoter regions, contained consensus transcription start sequences at both of the predicted promoters. S1 mapping with RNA prepared from maize endosperm showed that both P1 and P2 are active in vivo as double starts.

Electrophoretic elution of nucleic acids from gels adapted for subsequent biological tests: Application for analysis of mRNAs from maize endosperm

Gel electrophoresis has become a powerful tool for the analytical investigation of nucleic acid mixtures and for their preparative fractionation. However, the recovery of biologically active nucleic acids after electrophoresis has posed serious problems. The method of crushing the gel followed by elution often results in varying yields and partial loss of biological activity [ 1,2]. The method of electrophoretic elution of nucleic acids overcomes these problems. But, as applied so far, this method requires elaborate equip ment and skilled handling and can lead to dilute solutions [3-51. In all cases, the processing of several samples at the same time is rather time consuming. These disadvantages are overcome by the electrophoretic elution method described here. The procedure allows, without additional equipment, simultaneous elution of up to 20 samples/elution gel and has shown to yield, directly and with good recovery, biologically active mRNA, plasmid DNA and double-stranded DNA fragments. The performance of the method is demonstrated with mRNAs from maize endosperm and with DNA restriction fragments from a hybrid Col-plasmid.

Zein precursor mRNAs from maize endosperm

SummaryThe zein proteins are coded by a multigene family that is expressed exclusively in the endosperm of developing maize kernels. Both the proteins and their mRNAs are synthesized in large amounts. With improved methods of RNA preparation from endosperm it has been possible to detect high concentrations of several precursor forms of the zein mRNAs by Northern hybridizations with zein specific cDNA clones. A 1,500 base long precursor has also been identified by electron microscopy of R-loops formed between the precursor RNA and a maize genomic clone containing a zein gene. This genomic clone was also found to be active in germinal vesicle extracts from Xenopus oocytes producing transcripts corresponding in size to the final size zein mRNA and a larger, possible precursor mRNA.

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

SummaryAn EcoRI restriction endonuclease fragment of maize DNA coding for the 19,000 dalton zein protein was cloned in phage λgt 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.

Mapping of tissue-dependent and independent protein binding sites to the 5' upstream region of a zein gene

SummaryThe highly regulated expression of zein genes in endosperm tissue suggests that trans-acting factors, by binding to cis-acting sequences, influence the coordinate and developmentally regulated expression of these genes. A 15 55 bp 5′ flanking region of a zein gene was analysed for sites of specific interaction with nuclear proteins from endosperm and seedling tissue. At least four different protein binding sites were mapped to the zein 5′ region by the nitrocellulose filter binding technique and two of these exhibit tissue-specific binding.

Root-specific expression of a Zea mays gene encoding a novel glycine-rich protein, zmGRP3

The isolation and characterization of a cDNA clone from Zea mays coding for a novel glycine-rich protein (GRP) is described. The corresponding 1.4 kb mRNA accumulates exclusively in roots (primary, lateral seminal and crown roots) of young maize seedlings, following developmentally specific patterns. In agreement with previously described GRPs from other plant species the derived protein sequence exhibits a hydrophobic domain at the N-terminal region followed by repeated glycine-rich motifs. Genomic Southern analysis indicates that the zmGRP3 gene is present in the maize genome as one or two copies or at a low copy number.

Functional analysis of the — 300 region of maize zein genes

SummaryA 43 by fragment containing the — 300 region upstream control element common to the endosperm expressed zein genes of Zea mays L. has been analyzed by in vivo and in vitro techniques. Transient transformation studies with protoplasts from a maize endosperm culture indicate that the element positively affects CaMV 35S promoter-driven gene expression, and that this effect is both orientation- and position-dependent. Band-shift and Southwestern blotting experiments demonstrate that the element is specifically bound by different sets of DNA-binding proteins from seedling and endosperm nuclei. A 2 by substitution within the most conserved region of the element both reduces the stimulatory effect on transcription and alters the binding of nuclear proteins from both tissues.

Transcription from maize storage protein gene promoters in yeast.

Genes coding for zein storage proteins of maize of the 19 000‐ and 21 000‐dalton size classes were integrated into chromosome II of the yeast Saccharomyces cerevisiae. Using the transformed yeast strains as an in vivo transcription system it was shown that promoter regions of the zein genes of both size classes were accurately recognized. In particular, the zein mRNA synthesis in yeast starts at the same nucleotide positions used in the maize endosperm. A difference in the promoter activity between the 21 000‐ and the 19 000‐dalton zein genes was observed.