Transposon tagging of the maize Glossy2 locus with the transposable element En/Spm.

The Glossy2 (Gl2) locus of maize is required for the formation of the epicuticular wax layer of young plants. gl2 mutant seedlings can be visually identified because of their glossy leaf surface which is different from the dull surface of wild-type seedlings. The Gl2 locus was isolated by transposon tagging. Seven unstable mutations, gl2-m2 to gl2-m8, were induced in a parental strain carrying an active transposable Activator (Ac) element in the unstable wx-m7 allele. Genetic tests on the gl2-m2 allele indicated that it was not caused by the Ac element but by the insertion of the transposable element Enhancer/Suppressor-Mutator (En/Spm). A Sa/l restriction fragment segregating with the mutant phenotype was identified, by Southern analysis, using sequences from the En/Spm element as a probe. Part of the fragment was cloned and was shown to carry part of the unstable gl2-m2 allele. These gl2 sequences were used to identify a genomic fragment carrying the wild-type allele and to isolate its corresponding cDNA sequence. The predicted Glossy2 protein consists of 426 amino acids. No similar amino acid sequence was found in protein data banks and the biochemical function of the Gl2 gene product is still unknown. The wild-type Gl2 transcript is found predominantly in juvenile leaves. The transcript level in the leaves of seedlings homozygous for a stable recessive gl2-ref allele is hardly detectable.

Insertion mutations at the maize Opaque2 locus induced by transposable element families Ac, En/Spm and Bg.

Eight independently isolated unstable alleles of the Opaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-type O2 allele and the transposable element Activator (Ac) at the wx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of an Ac element. Unexpectedly, the remaining eight mutations were not caused by the designated Ac element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of the Enhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of the Bergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.

An unstable allele at the maize Opaque2 locus is caused by the insertion of a double Ac element.

An unstable allele designated o2-m55, was isolated as a derivative of the o2-m5 allele. Whereas the o2-m5 allele is caused by the insertion of one copy of the transposable element Activator (Ac) into the first exon of the maize Opaque2(O2) gene, the o2-m55 allele contains two Ac elements. The position of one copy is identical to the position of the Ac element in the o2-m5 allele. The second copy is present within the first copy, thereby interrupting its structure shortly before the first ATG of the major reading frame of Ac. Both Ac sequences have the same orientation. Excision of the internal Ac element as well as excision of the complete double Ac element was detectable. Truncated double Ac elements comprising the complete internal Ac element and either the proximal or distal fragment of the interrupted Ac element can also be excised. The Ac elements exhibit a strong negative dosage effect: kernels which display revertant sectors in a mutant background are rarely seen in plants homozygous for the o2-m55 allele. If only one dose of the o2-m55 allele is present in endosperm tissue, revertant sectors can be detected. The amount of the transcript expressed from the internal Ac element of the o2-m55 allele is less than that derived from the single Ac element present in the o2-m5 allele.

PCR-aided genomic sequencing of 5' subterminal sequences of the maize transposable element Activator (Ac) in transgenic tobacco plants.

The maize transposable element Activator (Ac) carries subterminal CpG-rich sequences which are essential for the transposition of the element. It has previously been shown that the methylation of certain sequences contained in this region can alter their ability to interact with the Ac-encoded protein. The novel hypothesis that the methylation of subterminal Ac sequences is required for transposition was tested. Approximately 150 bp of the 5' subterminal region of the Ac element was examined for the presence of 5-methylcytosines by the ligation-mediated polymerase chain reaction (LMPCR)-aided genomic sequencing method. The methylation status of 22 and 39 cytosines on either strand of the DNA were analysed in each of five different transgenic tobacco cultures carrying transposable Ac sequences. Ten micrograms of tobacco DNA were used for each base-specific cleavage reaction before amplification by LMPCR. All but one of the cytosines were unmethylated. Only a minor fraction of the Ac molecules was methylated at one cytosine residue. It is concluded that DNA methylation at the tested Ac sequences is not required for the transposability of Ac or Ds elements in tobacco cells.

Chromosomal rearrangements caused by the aberrant transposition of double Ds elements are formed by Ds and adjacent non-Ds sequences.

The Ds-induced unstable sh-m6258 allele is caused by the insertion of at least 45 kb of non-sh DNA. Genomic clones spanning the two junctions of sh sequences with insert sequences were isolated and analysed. The long insert in the sh-m6258 allele is bordered by Ds sequences on either side. A 3 kb double Ds structure which consists of one complete Ds element of approximately 2 kb and one half Ds element of approximately 1 kb was found in the sh-m6258 allele at the junction between the 3' region of the sh locus and the insert. At the junction between the 5' region of the sh locus and the insert a half Ds element is present. This truncated Ds element was probably left behind after the excision of a 2 kb Ds element from a 3 kb double Ds structure similar to that found at the 3' junction. Sequence analysis of the insert sequences beyond the Ds elements demonstrated that the long inserts in both the sh-m6258 and the sh-m5933 alleles originated from the same contiguous chromosomal segment in their common progenitor strain. One revertant derivative of the sh-m6258 allele was investigated. In the revertant strain, which displays a normal non-shrunken phenotype, a 2 kb Ds element is present at the site of the 45 kb insert in the mutant allele. This 2 kb insertion, which is located in the last but one intervening sequence of the Sh gene, does not inhibit expression of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Rare de novo methylation within the transposable element activator (Ac) in transgenic tobacco plants.

Transgenic SR1 tobacco plants that contained the maize transposable element Ac or deletion derivatives thereof were isolated. The DNA methylation patterns of the foreign DNA sequences were analysed with methylation-sensitive restriction enzymes. By this method we tested 87 cytosine residues whose methylation is known to inhibit restriction by the corresponding enzyme. Most of the restriction sites were cleavable and hence unmethylated in transgenic tobacco plants. There were only three restriction sites at which a fraction of the Ac sequences was methylated. A similar result was obtained with two inactive, internally or terminally deleted Ac sequences. In one of the deletion derivatives a single restriction site was completely methylated. All other sites were unmethylated. The complete Ac elements described in this report were present as single copies in the transgenic plants. Their activity was demonstrated by the presence of the element-specific transcript. The deletion derivatives did not transpose in the transgenic tobacco plants and were thus still linked to the sequences of the plasmid that was used for transformation. These adjacent sequences represent part of a chimaeric NPTII gene inactivated by the insertion of the Ac deletion derivative. All 16 restriction sites examined in this sequence were unmethylated.

Double Ds elements are involved in specific chromosome breakage.

The structure of the unstable Ds-induced sh-m5933 allele of the maize sucrose synthase gene was analysed and a double Ds structure found in opposite orientation on both sides of a 30 kb insert interrupting the sucrose synthase gene. The double Ds structures bordering the insert are identical over a distance of approximately 3 kb. These double Ds structures and the DNA segments beyond them are in opposite orientation and identical over a distance of approx. 5.3 kb. A hypothesis for how such a symmetrical structure could be formed is proposed. When one complete Ds element was excised from one of the double Ds structures a half Ds element was left behind. This half Ds element was found in one revertant strain which displayed an altered pattern of chromosome breakage compared to revertant strains which had not undergone Ds excision. Nine new maize strains which showed a similarly altered chromosome breakage pattern were isolated. In all nine cases we observed an indistinguishable deletion in the genomic DNA. These excisions are likely to be the result of similar excision events to that described above. We conclude that double Ds structures are responsible for Ds-induced chromosome breakage.

Analysis of sh-m6233, a mutation induced by the transposable element Ds in the sucrose synthase gene of Zea mays.

The unstable allele sh-m6233 caused by insertion of the transposable element Ds into the sucrose synthase gene of maize, was cloned. The mutation is caused by the insertion of an 4 kb DNA segment, consisting of two identical Ds elements of 2000 bp length, of which one is inserted into the center of the other in inverted orientation. This structure is, at the level of restriction mapping and partial DNA sequencing, identical to the double Ds element found in a larger insert in the mutant allele sh-m5933. 8 bp of host DNA are duplicated upon insertion. In a revertant, a 6-bp duplication is retained.

DNA sequence of the maize transposable element Dissociation.

The DNA sequence of the terminal 4.2 kilobases (kb) of the 30-kb insertion in the endosperm sucrose synthase gene of maize mutant sh-m5933 shows that it comprises two identical 2,040-base pair (bp) segments, one inserted in the reverse direction into the other. We suggest that the 2,040-bp sequence is an example of the transposable element Dissociation described by Barbara McClintock.

The controlling element Ds at the Shrunken locus in Zea mays: structure of the unstable sh-m5933 allele and several revertants.

We have analyzed the structure of the Shrunken (Sh) locus in a strain containing an unstable recessive mutation, sh-m5933, caused by the transposable controlling element Dissociation (Ds). We have also analyzed nine spontaneous Sh revertant alleles. The sh-m5933 allele contains a 30 kb insertion at the Sh locus, as well as a duplication that includes part of the insertion and the Sh locus sequence on the 5' side of the insertion site. The revertants continue to show Ds-mediated chromosome breakage at the Sh locus, have an intact Sh locus from which the insertion has been excised, and retain the duplication. One of the nine revertant alleles has a 2 kb deletion at the junction between the Sh locus and the insertion sequence in the duplicated segment of the locus. The revertant also shows a temporal change in the pattern of somatic chromosome breakage, implicating the junction sequence as the site of Ds-mediated chromosome breakage.

Genomic clones of a wild-type allele and a transposable element-induced mutant allele of the sucrose synthase gene of Zea mays L.

In an attempt to isolate the transposable genetic element Ds from Zea mays L., we cloned DNA fragments hybridizing to a cDNA clone derived from the sucrose synthase gene in a lambda vector (lambda::Zm Sh). The fragments cloned from wild-type and from the Ds-induced mutant sh-m5933 (lambda::Zm sh-m5933) share a segment 6 kb long while a contiguous segment of 15 kb of lambda::Zm sh-m5933 (mutant-derived DNA) does not hybridize to the DNA segment cloned from the wild-type. Restriction maps are given, and the junction point between the two DNA segments in the mutant clone was determined. Hybridization of DNA fragments, present in the wild-type DNA of lambda::Zm Sh, but not in the mutant clone, lambda::Zm sh-m5933, to genomic DNA of sh-m5933 showed that no part of this DNA is deleted. It cannot be said whether the DNA found in the mutant, but not in the wild-type clone, has been brought there by Ds insertion or by another Ds-dependent DNA rearrangement. The mutant-derived DNA was hybridized to genomic DNA of various maize lines digested by several restriction endonucleases. Approximately 40 bands were detected. The mutant-derived DNA contains two pairs of inverted repeats several hundred nucleotide pairs long, one of which is located at the junction to wild-type-derived DNA.

A cDNA clone from Zea mays endosperm sucrose synthetase mRNA.

A cDNA clone for maize endosperm sucrose synthetase of 62o nucleotide pairs length was obtained by cloning double stranded DNA obtained from the total maize endosperm poly(A) RNA in pBR322, and identifying the appropriate clone by hybrid-promoter translation. In Southern blotting to genomic BamHI-digested DNA, a single band only of approximately 20 Kb lights up, indicating that the sucrose synthetase gene is unique, or that closely linked copies are located on this DNA fragment.

DNA-methylase from regenerating rat liver: purification and characterisation.

DNA methylase has been purified 660-fold from nuclei from regenerating rat liver. The enzyme is able to methylate single stranded (ss) and double stranded (ds) DNA, the only reaction product being 5-methylcytosine. Previously unmethylated double stranded DNA from prokaryotes (M.luteus) as well as from eukaryotes (Ascaris suis) can serve as substrates. The synthetic copolymers (dG-dC)n . (dC-dG)n and (dG,dC)n are also methylated. While SV40 DNA is almost not methylated, PM2 DNA is a good substrate even in the supercoiled form. The enzyme methylates 1 in 17 bases in heterologous M.luteus DNA, but only 1 in 590 in homologous rat liver DNA. The high methylation level of M.luteus DNA, an analysis of the methylated pyrimidine isostichs and a preliminary dinucleotide analysis suggest that all the CpGs in a DNA can be methylated.