Jeff schell in cologne
Explore the source record for details and available documents.
Journal Article↗
Biomedical subjects
P Starlinger
Publications and source records attributed to P Starlinger.
The role of subterminal sites of transposable element Ds of Zea mays in excision.
Transposition depends on DNA sequences located at or near the termini of the transposon. In the maize transposable element Ds, these sequences were studied by site-directed mutagenesis followed by a transient excision assay in Petunia protoplasts. The transposase-binding AAACGG motifs found in large numbers in the element are important, but none of them is in itself indispensable, for excision. However, mutation of an isolated motif at the 3' end considerably reduced excisability. The inverted termini were confirmed to be indispensable. Point mutations in regions outside the inverted termini of Ds and not located in the transposase-binding motifs had, in some cases, a pronounced effect on excision frequency. The implications of these findings are discussed.
Base Sequence↗
What do we still need to know about transposable element Ac?
Transposable elements, originally discovered by Barbara McClintock, have been shown to occur in many if not all organisms. Their roles as selfish DNA (probable), as a major agent in evolution (unlikely) and as agents for the response to genomic stress (unclear) are discussed. Among the problems presently addressed are the mechanism of transposition and the regulation of transposition rate. The latter seems to differ in the Ac element of Zea mays compared to other transposable elements. The tendency of Ac transposase to form large aggregates is described, and the possible involvement of these aggregates in the control of the transposition rate is discussed.
Biological Evolution↗
Control of excision frequency of maize transposable element Ds in Petunia protoplasts.
The complete coding region of maize transposable element Ac and truncated but active derivatives of it were placed under the control of promoters of different strength and tested for the ability to excise transposable element Ds from a beta-glucuronidase reporter gene in a cotransfection assay in Petunia protoplasts. The highest excision values (5% of the protoplasts able to express the beta-glucuronidase gene in a control experiment) were observed with a truncated version of the Ac coding region under the control of the 2' promoter. The weak Ac promoter is sufficient to give rise to excision values not much lower than those found with much stronger promoters such as the 2' and nos promoters. A decrease in excision frequency was observed when translation of the Ac coding region was hindered by out-of-frame ATG codons in addition to the use of weak promoters. Increasing the level of Ac transposase thus does not seem to be sufficient to raise the level of Ds excision observed in this system and possibly also in maize. Therefore another factor limits the excision of Ds elements. Previously, it was reported that in tobacco cells the deletion of Ds sequence between base pairs 186 and 245 led to a decrease of the Ds excision frequency by the full-length but not by the truncated Ac product. In the Petunia assay system, however, deletion of these sequences decreased the excision rate with both the full length and the truncated Ac coding region. A cDNA construct was found similarly active as the corresponding genomic DNA.
Journal Article↗
Detection and abundance of mRNA and protein encoded by transposable element activator (Ac) in maize.
The 3.5 kb long mRNA of the maize transposable element Ac contains an open reading frame (ORFa) which encodes a polypeptide of 807 amino acids, the putative transposase of Ac. The Ac mRNA is a rare transcript: we now estimate the fraction of Ac mRNA in wx-m7::Ac seedlings to be 2-13 x 10(-5) of the polyA RNA. Assuming that maize cells contain similar amounts of polyA RNA as another monocot (0.16 pg/cell), this is equivalent to 1.5-10 transcripts in each cell. A protein with an apparent molecular weight of 112 kDa is detected, by five antisera directed against different segments of ORFa, exclusively in nuclear extracts from Ac-containing maize. This protein is most likely the full-length Ac ORFa protein. We estimate its concentration to be in the range of 3 x 10(-7) of the nuclear proteins, or about 1000 molecules per triploid endosperm cell containing one Ac element.
Blotting, Northern↗
Excision of a Ds-like maize transposable element (Ac delta) in a transient assay in Petunia is enhanced by a truncated coding region of the transposable element Ac.
The excision of a Ds-like transposable element (Ac delta) is mediated in trans by the transposable element Ac or its derivatives in Petunia protoplasts cotransfected with two plasmid DNAs. Excision restores the activity of the beta-glucuronidase (GUS) gene that is otherwise shut off by the presence of Ac delta in its leader sequence. A transient expression assay (histochemical test) is used to detect the beta-glucuronidase activity at the protoplast to detect the beta-glucuronidase activity at the protoplast level. The number of blue-stained protoplasts is a measure of the excision frequency. With Ac delta alone a near-zero background of GUS activity is detected, which is weakly enhanced by the presence, in trans, of either the wild-type Ac or the coding region (ORFa) transcribed from the 2' promoter of Agrobacterium tumefaciens TR-DNA. A strong enhancement is observed when a truncated Ac coding region, also under the control of the 2' promoter, is supplied in trans. The truncated version has ATG10 at codon 103 in frame with ORFa and is preceded by 7 out-of-frame ATGs. The assay is quick and well suited for detection of excision frequencies above the value obtained with the wild-type Ac. The presence of empty donor sites following excision can be demonstrated by PCR amplification and direct sequencing of the appropriate DNA fragment.
Base Sequence↗
Mutational analysis of the N terminus of the protein of maize transposable element Ac.
Mutations of transposable element Ac were tested for their capability to excise themselves from their location autonomously, to be excised by an active Ac, or to act in trans in the excision of an Ac delta element. Removal of 101 amino acids from the N terminus of the Ac protein does not decrease excision. A cis-acting site between base pairs 186 and 207 is important for excision by the wild-type protein but is not necessary for excision by the truncated protein. Improvement of the sequence context of the first AUG does not have a significant effect. Mutations in a small open reading frame of Ac encoding a 102-amino acid protein do not visibly alter excision frequency.
Base Sequence↗
The putative transposase of transposable element Ac from Zea mays L. interacts with subterminal sequences of Ac.
The Ac-specific ORFa protein, overexpressed in a baculovirus system, specifically binds to several subterminal fragments of Ac. The 11 bp long inverted repeats of the transposable element are not bound by the ORFa protein. Major ORFa protein-binding sites were delineated on 60 and 70 bp long sequence segments that lie 100 bp inside of the 5' Ac terminus and 40 bp inside of the 3' terminus respectively. Within all strongly bound fragments, and particularly in these 60 or 70 bp long segments, the hexamer motif AAACGG is repeated several times in direct or inverted orientation. The ORFa protein binds to synthetic concatemers of this motif, whereas the mutant motif AAAGGG is not complexed. Methylation of the cytosine residues in the AAACGG motif and/or its complementary strand has pronounced effects: whereas one of the two hemimethylated sequences has a higher affinity to the ORFa protein than both unmethylated and holomethylated DNAs, the other hemimethylated DNA is virtually not complexed at all. The native ORFa protein binding sites are more complex than the AAACGG sequence: certain Ac and Ds1 fragments devoid of AAACGG motifs (but containing several similar sequences) are weakly bound by the ORFa protein.
Base Sequence↗
Isolation and molecular analysis of the maize P locus.
The maize P locus is involved in the synthesis of a red flavonoid pigment in the pericarp, cob and other floral tissues. The tissue-specific pattern of expression of certain P alleles suggests that P may be a complex locus, with more than one functional unit. The P-VV allele, which specifies variegated pericarp and variegated cob, however, shows that insertion and excision of the transposable element Ac affects both pericarp and cob expression as though cob and pericarp pigmentation are controlled by a single gene. Using Ac as a transposon tag, we have isolated 34 kb of genomic DNA from the P-VV and P-RR allele. The cloned DNA contains two 5.8 kb cross-hybridizing regions, in direct orientation relative to each other, separated by 6.6 kb of intervening DNA. A sequence motif of 250 bp is repeated at three locations within the cloned region: once within each of the 5.8 kb repeats, and once outside the 5.8 kb repeats. DNA fragments flanking the Ac element detect five transcripts in RNA from wild type (P-RR) that are absent from mutant (P-VV) tissues. To localize the transcribed sequences, DNA probes spanning the 34 kb of cloned DNA were used in Northern analysis of RNA from mutant and wild-type kernels. The results suggest the presence of a single transcriptional unit located primarily within the DNA between the 5.8 kb repeats. The five RNAs transcribed from this region may be formed by alternative splicing. The size of the P gene derived from the length of the transcribed region seems much smaller than the gene size estimated from Ac-induced P-VV mutations.
Alleles↗
Sequences near the termini are required for transposition of the maize transposon Ac in transgenic tobacco plants.
Deletion derivatives of the maize transposable element Activator (Ac) were constructed in vitro and inserted into a kanamycin resistance gene. These constructions were then introduced into tobacco protoplasts derived from plants previously transformed with Ac. The ability of each deletion derivative to excise was measured by whether or not kanamycin-resistant tobacco calli were recovered. This allowed us to determine the length of DNA present at each terminus that is required to respond to the products expressed by the Ac element present in the genome. We show that around 200 base pairs (bp) are required at both ends for excision to occur at wild-type levels. When between 100 and 200 bp were retained at one of the ends, reduced frequencies of excision were detected. With less than 100 bp remaining at either end, no excision was detected. In addition, we show that although similar lengths of DNA are required at each terminus, the termini are not interchangeable. The significance of these data is discussed with respect to the protein(s) which interact(s) with the termini of Ac.
Blotting, Southern↗
Characterization of the maize transposable element Ac by internal deletions.
We have used the ability of Ac to transpose in tobacco to determine which Ac sequences are required for transposition, using a phenotypic assay for Ac excision from an NPTII gene in which excisions of Ac result in calli resistant to the antibiotic kanamycin (Baker et al., 1987). Here we show that deletion of the Ac DNA which encodes the untranslated leader of the Ac transcript does not prevent Ac excision. A deletion which removes 110 bp including the first 75 bp of long open reading frame prevents Ac excision in tobacco cells. However, it will excise in tobacco cells previously transformed with Ac indicating that deletion of the region prevents the synthesis of a product required for Ac excision. Deletion of the Ac sequences between bp 44 and bp 92 or from bp 75 to bp 181 abolishes, or strongly reduces, transposition in cells which are already transgenic for an active Ac element. This indicates that these deleted elements have lost sequences which are required for the transposon to respond to the transposase, when the enzyme is produced in trans. We also describe a tobacco strain transformed with a Ds element stably inserted wtihin an NPTII gene. This strain is Km and was retransformed with a construct containing the open reading frame (ORF) of the 3.5-kb Ac transcript expressed from a plant promoter. Expression of the cDNA construct promotes excision of the Ds element. These data suggest that the 3.5-kb transcript of Ac encodes the only Ac product required for transposition, i.e. the transposase function.
Journal Article↗
Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector.
The polypeptide encoded in the Activator (Ac) element of Zea mays L. has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPVs with the Ac-cDNA integrated and under the control of the viral polyhedrin promoter have been isolated and their genomes have been partly characterized as to the location of the foreign DNA insert. Upon infection of S. frugiperda cells with the recombinant AcNPV, maize Ac element specific messenger RNAs, as well as a newly synthesized polypeptide with an apparent molecular weight of about 116 kDa, have been detected in extracts of recombinant infected cells. This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies. Recombinant infected cells lack this protein. The Ac specific polypeptide is detected by antisera, which have been raised against fusion proteins containing Ac sequences synthesized in Escherichia coli, both in immunoprecipitation and in Western blotting experiments. The Ac specific protein is a nuclear phosphoprotein and represents about 1%-2% of the newly synthesized protein.
Animals↗
DNase I hypersensitive sites in the 5'-region of the maize Shrunken gene in nuclei from different organs.
The chromatin structure of the 5'-upstream region of the Shrunken (Sh) gene in Zea mays has been examined. We have identified a region of DNase I hypersensitivity extending at least from the 3'-end of exon 1 for 2 kb into the 5'-flanking region. This region is composed of a set of closely spaced hypersensitive sites separated by small regions that are less accessible to DNase I. The most sensitive sites are located within 300 bp upstream of the transcription start site. Hypersensitive sites are found essentially at the same positions in kernels, roots and leaves, although the latter display different relative intensities. No changes are found in roots within the tested region upon anaerobic induction. Testing protein-free plasmid DNA containing the 5' upstream region of the Sh gene, we found a site sensitive to the single strand specific nuclease S1 located very close to a DNase I hypersensitive site identified in chromatin. Several hypersensitive sites are flanking in vitro binding sites of nuclear proteins as determined by Werr et al. (1988; accompanying paper).
Base Sequence↗
Phenotypic assay for excision of the maize controlling element Ac in tobacco.
We describe a phenotypic assay designed to detect excision of the maize controlling element Ac from a selectable marker gene, neomycin phosphotransferase II (NPT II). An NPT II gene which expresses kanamycin resistance in tobacco cells, and contains a unique restriction enzyme site in the untranslated leader region, was constructed. Ac, or a defective Ac element (Ac big up tri, open), was inserted into the leader region of this gene. The transposon insertions inactivated the NPT II gene as determined by transient NPT II expression assays. The three plasmids were inserted into the T DNA of Agrobacterium tumefaciens Ti plasmid vectors, and transferred to tobacco protoplasts. The transformed protoplasts were selected with 100 or 200 microg/ml kanamycin. Protoplasts transformed by the NPT II gene interrupted by Ac formed 25% as many calli resistant to 100 or 200 microg/ml kanamycin as protoplasts transformed by the uninterrupted NPT II gene. Protoplasts transformed by the NPT II gene interrupted by Ac big up tri, open did not form any calli resistant to 200 microg/ml of kanamycin when transformed under similar conditions. Southern blot hybridization analyses of seven kanamycin-resistant calli or plants obtained after transformation by the NPT II gene interrupted by Ac revealed that in all cases Ac had excised, restoring the structure of the NPT II gene. This assay is therefore useful to monitor the activity of a transposable element such as Ac and to define the regions of this element involved in transposition activity.
Journal Article↗
Transcription of transposable element Activator (Ac) of Zea mays L.
Transcripts of various sizes hybridize to the transposable element Ac of Zea mays in most maize lines. A 3.5-kb mRNA with an abundance of 1-3 x 10 of the poly(A) RNA, however, is found exclusively in those lines that carry an active Ac. Plants with two Ac elements contain slightly more 3.5-kb Ac transcript than those with only one Ac. Overlapping cDNA clones spanning most of the message have been isolated and sequenced. The 5'-end of the transcript was determined by Northern hybridization and S1 mapping. It starts at several sites over a distance of nearly 100 bases, contains an AUG-free leader 600-700 nucleotides long, has a long open reading frame encoding 807 amino acids and an untranslated 3'-sequence of 239 nucleotides. Four introns with a combined length of 654 bases are removed from the primary transcript. Radiosequencing of in vitro translation products shows that translation of the long open reading frame begins at the first AUG, even though it is located in an unfavourable sequence context. The transcript is found in all organs investigated, provided an active Ac is present in the stock.
Journal Article↗
Cloning of the Bz2 locus of Zea mays using the transposable element Ds as a gene tag.
The Bz2 locus of Zea mays has been cloned, utilizing the presence of the transposable element Dissociation (Ds) at the locus as a gene flag. The Ds element inserted in the bz2-m allele was identified among many members of the Ac/Ds family in a Southern blot analysis of a population segregating for bz2-m and Bz2. After cloning a DNA fragment from the bz2-m allele, sequences flanking the Ds insertion were shown to be Bz2-specific and were used to isolate a homologous fragment from a wild-type Bz2 line. The Ds insertion in the bz2-m allele was found to be a Ds2 element identical to the Ds insertion in adh1-2F11.
Cloning, Molecular↗
Transposable element Ds2 of Zea mays influences polyadenylation and splice site selection.
In the allele adh1-2F11 of Zea mays the 1.3 kb transposable element Ds2 is inserted in the fourth exon of Adh1. Two major RNAs of 3.0 and 1.6 kb are transcribed from this allele. While the 3.0 kb transcript also contains the Ds2 sequences, the 1.6 kb transcript has lost the Ds2 sequences by an alternative splicing process. In this process, a normal 5' splice site is joined to a cryptic 3' splice site located within exon 4. This cryptic 3' splice site is not used in a wild-type Adh1F allele. Other minor transcripts of adh1-2F11 are prematurely terminated and polyadenylated.
DNA Transposable Elements↗
The Shrunken gene on chromosome 9 of Zea mays L is expressed in various plant tissues and encodes an anaerobic protein.
The Shrunken gene, located on the short arm of chromosome 9 of Zea mays, encodes the enzyme sucrose synthase (EC 2.4.1.13). The gene is known to be expressed in the endosperm of the developing maize kernel and seems to be involved in sucrose breakdown prior to starch synthesis. We have analyzed different tissues of the maize plant for transcripts of the Shrunken gene and have found rather high transcription rates in the etiolated shoot and the primary root of the germinating kernel. If the etiolated seedlings are illuminated, the transcript level drops by about 95% in the greening plant parts (1st and 2nd leaves) which are active in photosynthesis. A very low transcript level is found in mature green leaves where sucrose is formed from products of photosynthesis via a separate pathway. Upon anaerobic stress of the young seedling, the level of Shrunken transcripts increases 10 and 20 times in shoot and root tissue respectively. Apparently anaerobic induction supersedes the negative control that is observed after illumination in the 1st and 2nd leaves. From the experiments outlined here we conclude that the anaerobic protein 87 (ANP87, Hake et al. 1985) is encoded by the Shrunken locus. While the expression of the Shrunken gene varies in different tissues and in response to external stimuli, transcription of the second sucrose synthase (B) gene seems to be irresponsive to anaerobic stress and to be expressed at a similar low level in all of the tissues examined.
Alcohol Dehydrogenase↗