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R Kunze

Publications and source records attributed to R Kunze.

At least 19 recordsLinked to original sources
PubMed1997

Maize Activator transposase has a bipartite DNA binding domain that recognizes subterminal sequences and the terminal inverted repeats.

The mobility of maize transposable element Activator (Ac) is dependent on the 11-bp terminal inverted repeats (IRs) and approximately 250 subterminal nucleotides at each end. These sequences flank the coding region for the transposase (TPase) protein, which is required for the transposition reaction. Here we show that Ac TPase has a bipartite DNA binding domain, and recognizes the IRs and subterminal sequences in the Ac ends. TPase binds cooperatively to repetitive ACG and TCG sequences, of which 25 copies are found in the 5' and 20 copies in the 3' subterminal regions. TPase affinity is highest when these sites are flanked on the 3' side by an additional G residue (A/TCGG), which is found at 75% of binding sites. Moreover, TPase binds specifically to the Ac IRs, albeit with much lower affinity. Two mutations within the IRs that immobilize Ac abolish TPase binding completely. The basic DNA binding domain of TPase is split into two subdomains. Binding to the subterminal motifs is accomplished by the C-terminal subdomain alone, whereas recognition of the IRs requires the N-terminal subdomain in addition. Furthermore, TPase is extremely flexible in DNA binding. Two direct or inverted binding sites are bound equally well, and sites that are five to twelve bases apart are similarly well bound. The consequences of these findings for the Ac transposition reaction are discussed.

Base Sequence↗
PubMed1996

Binding sites for maize nuclear proteins in the subterminal regions of the transposable element Activator.

Genetic data suggest that transposition of the maize element Activator (Ac) is modulated by host factors. Using gel retardation and DNase I protection assays we identified maize proteins which bind to seven subterminal sites in both ends of Ac. Four DNase I-protected sites contain a GGTAAA sequence, the other three include either GATAAA or GTTAAA. The specificity of the maize protein binding to Ac was verified by using a synthetic fragment containing four GGTAAA motifs as probe and competitor in gel retardation assays. All seven binding sites are located within regions required in cis for transposition. A maize protein binding site with the same sequence has previously been identified in the terminal inverted repeats of the maize Mutator element. Thus, the protein, that recognizes this sequence is a good candidate for a regulatory host factor for Ac transposition.

Base Sequence↗
PubMed1996

Methylation pattern of Activator transposase binding sites in maize endosperm.

The maize transposable element Activator (Ac) transposes after replication from only one of the two daughter chromatids. It has been suggested that DNA methylation in conjunction with methylation-sensitive transposase binding to DNA may control the association of Ac transposition and replication. We present here a detailed genomic sequencing analysis of the cytosine methylation patterns of the transposase binding sites within both Ac ends in the wx-m9::Ac allele, where Ac is inserted into the tenth exon of the Waxy gene. The Ac elements in wx-m9::Ac kernels exhibit intriguing methylation patterns and fall into two distinct groups. Approximately 50% of the elements are fully unmethylated at cytosine residues through the 256 nucleotides at the 5' end (the promoter end). The other half is partially methylated between Ac residues 27 and 92. In contrast, at the 3' end, all Ac molecules are heavily methylated between residues 4372 and 4554. The more internally located Ac sequences and the flanking Waxy DNA are unmethylated. Although most methylated cytosines in Ac are in the symmetrical CpG and CpNpG arrangements, nonsymmetrical cytosine methylation is also common in the hypermethylated regions of Ac. These results suggest a model in which differential activation of transposon ends by hemimethylation controls the chromatid selectivity of transposition and the association with replication.

5-Methylcytosine↗
PubMed1995

The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize.

A reverse genetic system for studying excision of the transposable element Ds1 in maize plants has been established previously. In this system, the Ds1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of the Ac element, excision of Ds1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences required in cis for excision of Ds1. The Ds1 element contains the Ac transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5' subterminal region). We showed that mutation of these motifs abolished completely the excision capacity of Ds1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3') subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.

Base Sequence↗
PubMed1995

One of three nuclear localization signals of maize Activator (Ac) transposase overlaps the DNA-binding domain.

The nuclear localization sequences (NLSs) of the Ac transposase (TPase) protein have been characterized by indirect immunofluorescence detection of TPase deletion derivatives and TPase/beta-glucuronidase (GUS) fusion proteins in transiently transfected Petunia cells. The TPase contains three NLSs near its amino-terminal end, NLS(44-62), NLS(159-178) and NLS(174-206), each of which is sufficient to redirect GUS to the nucleus. Deletion of the N-terminal 102 TPase residues including NLS(44-62) results in strongly reduced nuclear import of the truncated TPase. NLS(44-62) and NLS(159-178) are bipartite NLSs, whereas the structure of NLS(174-206) does not allow a classification into one of the three major NLS categories. NLS(174-206) overlaps with the basic DNA-binding domain of TPase. A substitution of two amino acids in this segment (His191-->Arg and Arg193-->His) results in a total loss of DNA-binding activity, but retains reduced NLS activity. Accordingly, the two functions can be separated. In addition, we show that a NLS-deficient 71 kDa TPase derivative is co-imported into the nucleus in the presence of wild-type TPase.

Amino Acid Sequence↗
PubMed1995

[C-reactive protein (CRP)--an acute-phase protein with importance in laboratory medicine in veterinary medicine].

CRP is an acute phase protein (APP) formed by hepatic cells in acute phase reaction (APR). APR is initiated by a broad spectrum of cytokines of activated macrophages of blood and tissues. During the APR, plasma concentrations of APP are changed. Cytokines regulate APPs. CRP is a phylogenetically very old molecule. The degree of homology of amino acids in CRP is very high in a lot of species. CRP binding specificities depend on Ca++. The diagnostic importance of CRP in veterinary medicine is discussed.

Animals↗
PubMed1995

[C-reactive protein as a new parameter of mastitis].

The C-reactive protein (CRP) is an acute phase protein. Its deposit can be seen on several animal species. Following the development of a test system (enzyme-immunoassay/EIA) for the quantitative determination of bovine CRP, it was possible to detect this protein in bovine raw milk for the first time. It was shown in the first extensive investigations that the CRP concentration in raw milk was increased, sometimes as much as tenfold as a result of udder inflammation (mastitis). In several screening analyses of a herd of cows, the CRP-values were compared with the somatic cell number, fat, protein and lactose content. The degree of CRP concentrations in raw milk in cows suffering from mastitis during the antibiotic treatment was reflected in the treatment success. When CRP was used as an additional parameter of selection, a better average udder health could be seen after five months. The first results of our investigations demonstrate that CRP is a practicable indicator of inflammation and may be effectively used to determine and control udder illness in cows.

Animals↗
PubMed1995

[Density of adhesive proteins after oral administration of proteolytic enzymes in multiple myeloma].

The authors present information on the presence of adhesive proteins on membranes of myeloma and precursor cells isolated from bone marrow and blood from a group of 33 patients examined by fluorescent flow cytometry. They also compare the density of integrins (CD29, CD49e, CD41, CD51 and CD61) and adhesive proteins from the group of "homing" receptors (CD44) and IgG "superfamily" (LFA-1, LFA-3, ICAM-1, N-CAM) and their changes after a single oral dose of a mixture of proteolytic enzymes (Wobe Mugos, Wobenzym, MUCOS Pharma, FRG). The authors observed a significant drop of CD29, CD54 (ICAM-1), CD58 (LFA-3) after Wobe Mugos, CD49, CD51, CD58 after Wobenzyme. The insignificant decline of density of CD44 on cells, as well as of the soluble receptor of CD44 after oral administration of proteolytic enzymes in serum, incl. the mentioned changes of integrins and other adhesive proteins, indicate the importance of enzyme preparations in the supporting treatment of malignant processes.

Adjuvants, Immunologic↗
PubMed1994

In vivo aggregation of maize Activator (Ac) transposase in nuclei of maize endosperm and Petunia protoplasts.

The transposase (TPase) of the maize transposon Activator (Ac) accumulates in the nuclei of maize endosperm and transfected Petunia protoplasts, where it aggregates into rod-like structures about 2 microm in length. In petunia protoplasts the amount of TPase aggregates increases with the strength of the promoter fused to the Ac-coding region. The excision frequency of a Ds element, however, does not increase proportionally. The data suggest that the aggregated TPase is not responsible for the mobilization of the Ds element, but rather is a transpositionally inactive form of the protein. In contrast to the full-length TPase, a functional, N-terminally truncated TPase derivative is inefficiently transported into the nucleus at high expression levels and aggregates predominantly in the cytoplasm. Accordingly, the N-terminus of the TPase is involved in nuclear localization and/or aggregation.

Amino Acid Sequence↗
PubMed1993

Dominant transposition-deficient mutants of maize Activator (Ac) transposase.

The maize transposable element Activator (Ac) encodes a transposase (TPase) protein, whose DNA-binding domain is located in a basic region around aa 200. The N-terminal 102 aa of the TPase are not required for the transposition reaction. In transfected petunia protoplasts, we analyzed the protein levels of the N-terminally truncated TPase and mutants thereof and the corresponding transposition frequencies. The TPase protein forms large insoluble aggregates at high expression levels. There is no proportionality observed between TPase levels and transposition frequency. Twenty-one mutations (of 26), which are distributed over the whole length of the protein, inactivate the TPase completely. By coexpressing inactive mutant and active truncated TPase, it was found that several mutations have a trans-dominant inhibitory effect. Among those are two DNA-binding-deficient mutants, indicating that inhibition of the active TPase is not caused by competition for the binding sites on the transposon. Accordingly, Ac TPase acts as an oligo- or multimer formed by protein-protein interactions. Peculiarly, two mutants lacking 53 and 98 aa from the C terminus that are themselves transpositionally inactive lead to an increased excision frequency when they are coexpressed with the active truncated TPase.

Amino Acid Sequence↗
PubMed1992

High rates of Ac/Ds germinal transposition in Arabidopsis suitable for gene isolation by insertional mutagenesis.

Overexpression of the Activator (Ac) transposase gene in Arabidopsis thaliana resulted in a minimal germinal transposition frequency of 27% in which independent Dissociation (Ds) transposition events were observed. Molecular analysis of 45 F1 generation Ac/Ds plants indicated that high rates of somatic excision had occurred, and independent germinal insertions were identified in F2 generation progeny plants. A tandem cauliflower mosaic virus (CaMV) promoter fused to two different Ac coding sequences significantly increased the rate of Ds transposition. The CaMV-Ac fusions activated single and multiple copies of two different Ds elements, DsDHFR and Ds35S-1, and reciprocal crosses resulted in similar transposition frequencies. The improved rate of independent germinal transposition observed makes Arabidopsis an ideal system for insertional mutagenesis.

Base Sequence↗
PubMed1992

Immunocytochemical detection of herpes viruses in oral smears of HIV-infected patients.

Cytologic smears (CS) were taken from the lateral border of the tongue of HIV-seropositive patients (HIV+) (n = 39) and of seronegative controls (HIV-) (n = 19) and examined by immunocytochemistry (APAAP) and in situ hybridization (ISH) (biotinylated DNA probes) for the presence of viral antigens/DNA of EBV and CMV. While none of the HIV controls showed positive results for EBV antigen, 61% (APAAP) resp. 79% (ISH) of oral epithelial cells in the group of HIV+ patients were EBV-positive. While all CS taken from areas with the clinical diagnosis of hairy leukoplakia (HL) were EBV positive (APAAP and/or ISH), the detection of EBV in CS from uninvolved oral mucosa seemed to be associated with the later development of HL. In the group of HIV+ patients the detection rate for CMV was about five times (APAAP) resp. three times (ISH) higher than in HIV- persons. This non-invasive technique seems to be a valuable tool to screen for viral antigens/genomes.

Adult↗
PubMed1992

Distribution of cytokeratins in oral cytological smears of HIV-infected patients.

Cytological smears (CS), taken from the lateral border of the tongue of HIV-seropositive patients (HIV+) (n = 34) and of seronegative controls (HIV-) (n = 16), were examined by means of immunocytochemistry (APAAP) for the distribution patterns of different cytokeratins and MHC class II antigens. Compared with HIV- patients in CS of HIV-infected patients cornification associated cytokeratins 10/11 were increased, while the number of keratinocytes positive for cytokeratins 13/16 was comparable in both groups. Expression of simple epithelial cytokeratins 19, rarely observed in CS of HIV- patients, was a frequent findings in CS of HIV+ patients. Keratinocytes positive for MHC class II antigens were observed in CS of 12/34 HIV+, while all control CS were negative. In the group of HIV+ patients no correlation was found between the clinical presence of HL and the expression of cytokeratins or class II antigens. The altered distribution of cytokeratins may reflect local responses to proliferative stimuli or local inflammation due to the presence of microbial antigens or may occur as a general unspecific reaction in the setting of systemic viral infection. This non-invasive technique seems to be a valuable tool to determine the proliferation rate of oral epithelial cells.

Adult↗
PubMed1991

The ORFa protein, the putative transposase of maize transposable element Ac, has a basic DNA binding domain.

Ac encodes the 807 amino acid ORFa protein which binds specifically to multiple AAACGG motifs that are subterminally located in both ends of Ac. The wild-type ORFa protein and a number of deletion and amino acid exchange mutants were expressed in Escherichia coli, renatured and used for mobility shift assays. At least 136 amino acids from the N-terminus and 537 C-terminal amino acids may be removed from the ORFa protein without destroying the DNA binding domain, whereas a protein starting at amino acid 189 is DNA binding deficient. Certain basic amino acids between positions 190 and 200 are essential for DNA binding, as their substitution with uncharged amino acids leads to the loss of this function. The DNA binding domain of ORFa protein has an overall basic character, but no obvious sequence homology to any other known DNA binding protein. The homologies to the major open reading frames of transposable elements Tam3 from Antirrhinum majus and Hobo from Drosophila are found between the C-terminal two thirds of the three proteins. The ORFa protein forms discrete complexes with target DNA that appear, depending on the protein concentration, as a 'ladder' of bands on gels, indicating the occupation of target DNA by multiple ORFa protein molecules.

Amino Acid Sequence↗
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↗