Peter A. Peterson

The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators.

The structure of the wild‐type c1 locus of Zea mays was determined by sequence analysis of one genomic and two cDNA clones. The coding region is composed of three exons (150 bp, 129 bp and one, at least 720 bp) and two small introns (88 bp and 145 bp). Transcription of the mRNAs corresponding to the two cDNA clones cLC6 (1.1 kb) and cLC28 (2.1 kb) starts from the same promoter. Both cDNAs are identical except that cLC28 extends further at its 3′ end. A putative protein, 273 amino acids in length was deduced from the sequence of both transcripts. It contains two domains, one basic and the other acidic and might function as a transcriptional activator. The basic domain of this c1‐encoded protein shows 40% sequence homology to the protein products of animal myb proto‐oncogenes.

Molecular cloning of the a1 locus of Zea mays using the transposable elements En and Mu1.

The a1 locus of Zea mays has been cloned using transposable elements as gene tags. The strategy was to make genomic libraries from maize stocks with a1 mutations induced either by En(Spm) or by Robertsons Mutator‐system. These libraries were then screened with either Spm‐I8 and En1, for the En‐containing mutant, or with Mu1 for the Mu‐induced mutation. There are many En and Mu1 hybridizing sequences present in the maize genome, however, by a process of cross‐screening of the positives from the two libraries and by molecular analysis of the En‐positive clones it was possible to identify clones in both libraries carrying all or part of the a1 gene.

Influence of transposable elements on the structure and function of the A1 gene of Zea mays

The structure of the A1 gene of Zea mays was determined by sequencing cDNA and genomic clones. The gene is composed of four exons and three short introns. The 40.1‐kd A1 protein is an NADPH‐dependent reductase. Germinal derivatives of the mutable a1‐ml allele with either recessive or wild‐type phenotype have been isolated. Sequence analysis of these revertant alleles indicates that frame‐shift mutations abolish A1 gene function, whereas one additional amino acid within the protein sequence still allows wild‐type gene expression. The presence of a second, promoter‐like structure, upstream of the functional A1 gene promoter is discussed with respect to its possible involvement in differential expression of the A1 gene. The structure of the a1‐m2 8004, 3456 and 4412 alleles, featuring distinguishable phenotypes in the presence of Spm(En), was also determined. In all alleles the 1080‐bp‐long inhibitor (I) element is located 15 bp upstream of the CAAT box of the A1 gene promoter. The unusual response of al‐m2 alleles to trans‐active signals of the Spm(En) element is discussed with respect to the position of the I inserts. Also presented are data on the structure and insertion sites of transposable elements determined by cloning and sequencing of the mutable a1 alleles a1‐mpapu, a1‐mr 102 and al‐ml.

Molecular cloning of the c2 locus of Zea mays, the gene coding for chalcone synthase

SummaryThe c2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable element En (Spm) as a gene tag. The Spm element present at the c2 locus in the autonomously mutating c2-m1 line was isolated using En1 element specific probes. Sequences flanking the element were identified as c2 locus specific and were used to clone the nonautonomous c2-m2 and wild-type alleles. The cloning and analysis of a cDNA complementary to the c2 locus provided evidence that this gene encodes the enzyme chalcone synthase.

Molecular cloning of the c locus of Zea mays: a locus regulating the anthocyanin pathway

The c locus of Zea mays, involved in the regulation of anthocyanin biosynthesis, has been cloned by transposon tagging. A clone (#18En) containing a full size En1 element was initially isolated from the En element‐induced mutable allele c‐m668655. Sequences of clone #18En flanking the En1 element were used to clone other c mutants, whose structure was predicted genetically. Clone #23En (isolated from c‐m668613) contained a full size En1 element, clone #3Ds (isolated from c‐m2) a Ds element and clone #5 (isolated from c+) had no element on the cloned fragment. From these data we conclude that the clones obtained contain at least part of the c locus. Preliminary data on transcript analysis using a 1‐kb DNA fragment from wild‐type clone #5 showed that at least three transcripts are encoded by that part of the locus, indicating that c is a complex locus.

The En/Spm transposable element of Zea mays contains splice sites at the termini generating a novel intron from a dSpm element in the A2 gene.

The A2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable elements rcy and dSpm as gene tags. The A2 gene encodes a putative protein of 395 amino acids and is devoid of introns. Two a2‐m1 alleles, containing dSpm insertions of different sizes, were characterized. The dSpm element from the original state allele has perfect termini and undergoes frequent transposition. The element from the class II state allele is no longer competent to transpose. It has retained the 13 bp terminal inverted repeat but has lost all subterminal sites at the 5′ end, which are recognized by tnpA protein, the most abundant product of the En/Spm transposable element system. The relatively high A2 gene expression of one a2‐m1 allele is due to removal of almost all dSpm sequences by splicing. The slightly altered A2 enzyme is still functional as shown by complementation of an a2 mutant with the corresponding cDNA. The 5′ and 3′ splice sites are constituted by the termini of the dSpm element; it therefore represents a novel intron of the A2 gene.

Genetic and molecular analysis of the Enhancer (En) transposable element system of Zea mays.

A newly isolated, unstable mutation wx‐844::En‐1 of Zea mays was proven to be caused by the insertion of the autonomous transposable element En into the Waxy (Wx) gene. Molecular analysis revealed that En‐1 is 8.4 kb long, has a 13‐bp long perfect inverted repeat at its termini and generates a 3‐bp target site duplication. En‐1 is integrated into an intron located approximately in the middle of the transcribed region of the Wx gene. Structural evidence is presented indicating that a receptor component (Inhibitor) can arise by internal deletion of an autonomous En element.

A general method to identify plant structural genes among genomic DNA clones using transposable element induced mutations

SummarySeveral genomic clones from Petroselinum hortense, Zea mays and Antirrhinum majus all homologous to cloned Petroselinum chalcone synthase cDNA were isolated using the λgt WES cloning system.Clones containing the chalcone synthase structural gene were identified by hybridization to cDNA from Petroselinum hortense, genomic wildtype, mutant and revertant DNA.Among the 5 different clones from Petroselinum hortense, PH3 is the most likely candidate to contain at least a portion of the chalcone synthase gene.None of the 4 Zea mays clones appeared to contain part of the chalcone synthase gene.Among the 2 different clones from Antirrhinum majus, AM3 contains the portion of the chalcone synthase structural gene which is altered in the mutant nivea recurrens (nivrec). This mutant is considered to be due to the integration of a transposable element. In revertants of nivrec to niv+ the wildtype locus is restored molecularly.

The En mutable system in maize. III. Transposition associated with mutational events.

Summary1.The mutable allele, a1m(pa−pu) of the En system at the a1 locus in maize mutates somatically and germinally to pale, colorless, and purple.2.Colorless and pale germinal deviants arise at a high frequency. The colorless is more frequent than the pale, and each is more frequent than purple. Frequency is correlated with timing of the somatic mutation event — the earlier colorless sectoring is correlated with the higher frequency of colorless deviants.3.The regulatory element, En, has been identified at the a1 locus. The origin of colorless and pale deviants is accompanied by the transposition of an En element away from the a1 site.4.The transposing event may lead to implantation of En on the same chromosome, on another chromosome, or no implantation occurs. Transposition to a linked site occurs approximately 25% of the time. There is a preference for transposition to sites 6–20 units from a1.5.Secondary transpositions of En occur, and in one test, approximately 12% of the time, to an independent position. Secondary transpositions take place to new linked sites.6.Preliminary data indicate that transpositions can occur to both distal and proximal positions on chromosome 3.7.Since differences exist in the behaviour of elements in transposition, it is likely that the transposition event probably is dependent on the elements of specific mutable systems and differing elements within a system.8.Theoretical aspects of diverse types of impairment of normal gene function by inserted elements is discussed.

Mobile elements in plants

Before beginning this review of transposable elements in plants, a few comments on general concepts are appropriate. The collective feature that encompasses this review is variegation in plants (Figure 1 A, B, C, and D). This variegation has been tied to genetic elements (sequences of small pieces of DNA) intimately integrated into genes, whose mobility (transposition) in and out of genes yields the variegated phenotype. Any particular variegation that one sees, e.g., the colored spots in Figure 1A or in Figure 10, is the outward expression (phenotype) of the mobility (excision) of these transposing elements, and for any one variegated phenotype it is one particular element in one gene. Without this association of the element with a specific gene that monitors the element, variegation would not be obvious. Any gene could have one of these elements integrated and any one of many different kinds (systems) of elements could be integrated into any one gene. Thus far, no gene is known to be immune to “visitati...