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P Starlinger

Publications and source records attributed to P Starlinger.

At least 19 recordsLinked to original sources
PubMed1991

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1986

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↗
PubMed1979

IS4 is found between eleven or twelve base pair duplications.

Three mutations caused by the integration of IS4 in galT in both possible orientations were shown by DNA sequence analysis to be integrated between a duplication of eleven base pairs of gene galT. IS4 has been cloned from its single position on the E. coli K12 chromosome. Here, 12 base pairs are duplicated adjacent to IS4. This sequence is unrelated to the duplicated sequence in galT.

Base Sequence↗
PubMed1979

Close vicinity of IS1 integration sites in the leader sequence of the gal operon of E. coli.

Four insertions of IS1 in the leader sequence of the gal operon of E. coli have been analysed. Two of them occur at the same position, but in opposite orientations. The other two are inserted one nucleotide to one side and four nucleotides to the other side, respectively. In each case, nine base pairs of the leader sequence of the gal operon are duplicated directly, and are found flanking the termini of IS1 at its junction with the gal operon. These repeated sequences differ from each other as expected from the different insertion sites.

Base Sequence↗
PubMed1979

Nomenclature of transposable elements in prokaryotes.

Transposable elements are defined as specific DNA segments that can repeatedly insert into a few or many sites in a genome. They are classified as simple IS elements, more complex Tn transposons and self-replicating episomes. Definitions and nomenclature rules for these three classes of prokaryotic transposable elements are specified.

Cells↗