Fischartbestimmung von Caviar durch Protein- und DNA-Analyse

Deutscher Caviar, made from roe of lumpfish or capelin, gives species specific patterns in protein electrophoresis. The same techniques can be used to differentiate caviar from salmon and trout. The differentiation of sturgeon caviar (beluga, osietra, sevruga) is possible by isoelectric focusing, but not by SDS-PAGE. PCR-based methods of DNA-analysis for identification of the origin of sturgeon caviar are under development.

New DNA based hybrid catalysis

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Sequence specific recognition and cleavage of DNA by synthetic molecules

DNA recognition is an essential biological process responsible for the regulation of cellular functions including protein synthesis and cell division and is implicated in the mechanism of action of some anticancer drugs. Studies directed towards defining the elements responsible for sequence specific DNA recognition through the study of the interactions of synthetic organic ligands with DNA are described.

DNA recognition by poly-N-methylpyrrolecarboxamides was studied by the synthesis and characterization of a series of molecules where the number of contiguous N-methylpyrrolecarboxamide units was increased from 2 to 9. The effect of this incremental change in structure on DNA recognition has been investigated at base pair resolution using affinity cleaving and MPE•Fe(II) footprinting techniques. These studies led to a quantitative relationship between the number of amides in the molecule and the DNA binding site size. This relationship is called the n + 1 rule and it states that a poly-N methylpyrrolecarboxamide molecule with n amides will bind n + 1 base pairs of DNA. This rule is consistent with a model where the carboxamides of these compounds form three center bridging hydrogen bonds between adjacent base pairs on opposite strands of the helix. The poly-N methylpyrrolecarboxamide recognition element was found to preferentially bind poly dA•poly dT stretches; however, both binding site selection and orientation were found to be affected by flanking sequences. Cleavage of large DNA is also described.

One approach towards the design of molecules that bind large sequences of double helical DNA sequence specifically is to couple DNA binding subunits of similar or diverse base pair specificity. Bis-EDTA-distamycin-fumaramide (BEDF) is an octaamide dimer of two tri-N methylpyrrolecarboxamide subunits linked by fumaramide. DNA recognition by BEDF was compared to P7E, an octaamide molecule containing seven consecutive pyrroles. These two compounds were found to recognize the same sites on pBR322 with approximately the same affinities demonstrating that fumaramide is an effective linking element for Nmethylpyrrolecarboxamide recognition subunits. Further studies involved the synthesis and characterization of a trimer of tetra-N-methylpyrrolecarboxamide subunits linked by β-alanine ((P4)_(3)E). This trimerization produced a molecule which is capable of recognizing 16 base pairs of A•T DNA, more than a turn and a half of the DNA helix.

DNA footprinting is a powerful direct method for determining the binding sites of proteins and small molecules on heterogeneous DNA. It was found that attachment of EDTA•Fe(II) to spermine creates a molecule, SE•Fe(II), which binds and cleaves DNA sequence neutrally. This lack of specificity provides evidence that at the nucleotide level polyamines recognize heterogeneous DNA independent of sequence and allows SE•Fe(II) to be used as a footprinting reagent. SE•Fe(II) was compared with two other small molecule footprinting reagents, EDTA•Fe(II) and MPE•Fe(II).

Regenerative amplifier with continuously variable pulse duration used in an optical parametric chirped-pulse amplification laser system for synchronous pumping

A Nd:glass regenerative amplifier has been set up to generate the pumping pulse with variable pulse width for an optical parametric chirped-pulse amplification (OPCPA) laser system. Each pulse of the pulse train from a cw self-mode-locking femtosecond Ti:sapphire oscillator is stretched to approximate to300 ps at 1062 nm to be split equally and injected into a nonlinear crystal and the Nd:glass regenerative amplifier, as the chirped signal pulse train and the seed pulse train of the pumping laser system, respectively. By adjusting the cavity length of the regenerative amplifier directly, the width of amplified pulse could be varied continuously from approximate to300 ps to approximate to3 ns. The chirped signal pulse for the OPCPA laser system and the seed pulse for the pumping laser system come from the same oscillator, so that the time jitter between the signal pulse and the pumping pulse in optical parametric amplification stages could be <10 ps. (C) 2003 Society of Photo-Optical Instrumentation Engineers.

Temporal synchronization in optical parametric chirped pulse amplification laser system

The effect of temporal synchronization between the chirped signal pulse and the pumping pulse in an optical parametric chirped pulse amplification laser system is researched theoretically and experimentally. The results show that the gain of optical parametric amplification is sensitive to the temporal synchronization. Therefore, accurate temporal synchronization between the chirped signal pulse and the pumping pulse is essential to obtain high optical parametric amplification gain and stable output from an optical parametric chirped pulse amplification laser. Based on our 16.7-TW/120-fs optical parametric chirped pulse amplification laser system with similar to1-ns pumping pulse duration and <10-ps time jitter between the signal and pumping pulse, the effect of the temporal synchronization on optical parametric chirped pulse amplification is demonstrated. The experimental results agree with the calculation. (C) 2004 Society of Photo-Optical Instrumentation Engineers.

Broadband high-gain optical parametric chirped-pulse amplification near 780 nm with LBO-I near-degenerate near-collinear phase match

Near-degenerative near-collinear phase-match geometry for broadband optical parametric chirped-pulse amplification (OPCPA) at approximate to 780 nm is calculated in comparison with nondegenerate noncollinear phase-match geometry. In an experiment on LBO-I near-degenerate near-collinear OPCPA, high gain with broad gain bandwidth (approximate to 71 nm, FWHM) at approximate to 780 nm is achieved by using an approximate to 390-nm pumping pulse. The stretched broadband chirped signal pulse near 780 nm is amplified to approximate to 412 mu J with a pumping energy of approximate to 15 mJ, and the total gain is > 3.7 X 10(6), which agrees well with the calculation. For a broadband (covering approximate to 100 nm) chirped signal pulse, the theoretical gain bandwidth has been attained experimentally for the first time. (c) 2005 Society of Photo-Optical Instrumentation Engineers.

Multiterawatt laser system based on optical parametric chirped pulse amplification

A compact multiterawatt laser system based on optical parametric chirped pulse amplification is demonstrated. Chirped pulses are amplified from 20 pJ to 900 mJ by two lithium triborate optical parametric preamplifiers and a final KDP optical parametric power amplifier with a pump energy of 5 J at 532 nm from Nd:YAG-Nd: glass hybrid amplifiers, After compression, we obtained a final output of 570-mJ-155-fs pulses with a peak power of 3.67 TW, which is the highest output power from an optical parametric chirped pulse amplification laser, to the best of our knowledge. (C) 2002 Optical Society of America.

Broadband high-gain optical parametric chirped-pulse amplification near 780 nm with LBO-I near-degenerate near-collinear phase match

Near-degenerative near-collinear phase-match geometry for broadband optical parametric chirped-pulse amplification (OPCPA) at approximate to 780 nm is calculated in comparison with nondegenerate noncollinear phase-match geometry. In an experiment on LBO-I near-degenerate near-collinear OPCPA, high gain with broad gain bandwidth (approximate to 71 nm, FWHM) at approximate to 780 nm is achieved by using an approximate to 390-nm pumping pulse. The stretched broadband chirped signal pulse near 780 nm is amplified to approximate to 412 mu J with a pumping energy of approximate to 15 mJ, and the total gain is > 3.7 X 10(6), which agrees well with the calculation. For a broadband (covering approximate to 100 nm) chirped signal pulse, the theoretical gain bandwidth has been attained experimentally for the first time. (c) 2005 Society of Photo-Optical Instrumentation Engineers.

Dispersion analysis and compensation of collinear optical parametric chirped pulse amplification systems

In an optical parametric chirped pulse amplification (OPCPA) laser system, residual phase dispersion should be compensated as much as possible to shorten the amplified pulses and improve the pulse contrast ratio. Expressions of orders of the induced phases in collinear optical parametric amplification (OPA) processes are presented at the central signal wavelength to depict a clear physics picture and to simplify the design of phase compensation. As examples, we simulate two OPCPA systems to compensate for the phases up to the partial fourth-order terms, and obtain flat phase spectra of 200-nm bandwidth at 1064 nm and 90-nm at 800 nm.

Recognition of double helical DNA by purine oligonucleotides via triple helix formation

Oligonucleotide-directed triple helix formation is one of the most versatile methods for the sequence specific recognition of double helical DNA. Chapter 2 describes affinity cleaving experiments carried out to assess the recognition potential for purine-rich oligonucleotides via the formation of triple helices. Purine-rich oligodeoxyribonucleotides were shown to bind specifically to purine tracts of double helical DNA in the major groove antiparallel to the purine strand of the duplex. Specificity was derived from the formation of reverse Hoogsteen G•GC, A•AT and T•AT triplets and binding was limited to mostly purine tracts. This triple helical structure was stabilized by multivalent cations, destabilized by high concentrations of monovalent cations and was insensitive to pH. A single mismatched base triplet was shown to destabilize a 15 mer triple helix by 1.0 kcal/mole at 25°C. In addition, stability appeared to be correlated to the number of G•GC triplets formed in the triple helix. This structure provides an additional framework as a basis for the design of new sequence specific DNA binding molecules.

In work described in Chapter 3, the triplet specificities and required strand orientations of two classes of DNA triple helices were combined to target double helical sequences containing all four base pairs by alternate strand triple helix formation. This allowed for the use of oligonucleotides containing only natural 3'-5' phosphodiester linkages to simultaneously bind both strands of double helical DNA in the major groove. The stabilities and structures of these alternate strand triple helices depended on whether the binding site sequence was 5'-(purine)_m (pyrimidine)_n-3' or 5'- (pyrimidine)_m (purine)_n-3'.

In Chapter 4, the ability of oligonucleotide-cerium(III) chelates to direct the transesterfication of RNA was investigated. Procedures were developed for the modification of DNA and RNA oligonucleotides with a hexadentate Schiff-base macrocyclic cerium(III) complex. In addition, oligoribonucleotides modified by covalent attachment of the metal complex through two different linker structures were prepared. The ability of these structures to direct transesterification to specific RNA phosphodiesters was assessed by gel electrophoresis. No reproducible cleavage of the RNA strand consistent with transesterification could be detected in any of these experiments.

Design, synthesis and characterization of sequence-specific DNA-cleaving metalloproteins

This thesis describes research pursued in two areas, both involving the design and synthesis of sequence specific DNA-cleaving proteins. The first involves the use of sequence-specific DNA-cleaving metalloproteins to probe the structure of a protein-DNA complex, and the second seeks to develop cleaving moieties capable of DNA cleavage through the generation of a non-diffusible oxidant under physiological conditions.

Chapter One provides a brief review of the literature concerning sequence-specific DNA-binding proteins. Chapter Two summarizes the results of affinity cleaving experiments using leucine zipper-basic region (bZip) DNA-binding proteins. Specifically, the NH_2-terminal locations of a dimer containing the DNA binding domain of the yeast transcriptional activator GCN4 were mapped on the binding sites 5'-CTGACTAAT-3' and 5'ATGACTCTT- 3' using affinity cleaving. Analysis of the DNA cleavage patterns from Fe•EDTA-GCN4(222-281) and (226-281) dimers reveals that the NH_2-termini are in the major groove nine to ten base pairs apart and symmetrically displaced four to five base pairs from the central C of the recognition site. These data are consistent with structural models put forward for this class of DNA binding proteins. The results of these experiments are evaluated in light of the recently published crystal structure for the GCN4-DNA complex. Preliminary investigations of affinity cleaving proteins based on the DNA-binding domains of the bZip proteins Jun and Fos are also described.

Chapter Three describes experiments demonstrating the simultaneous binding of GCN4(226-281) and 1-Methylimidazole-2-carboxamide-netropsin (2-ImN), a designed synthetic peptide which binds in the minor groove of DNA at 5'-TGACT-3' sites as an antiparallel, side-by-side dimer. Through the use of Fe•EDTA-GCN4(226-281) as a sequence-specific footprinting agent, it is shown that the dimeric protein GCN4(226-281) and the dimeric peptide 2- ImN can simultaneously occupy their common binding site in the major and minor grooves of DNA, respectively. The association constants for 2-ImN in the presence and in the absence of Fe•EDTA-GCN4(226-281) are found to be similar, suggesting that the binding of the two dimers is not cooperative.

Chapter Four describes the synthesis and characterization of PBA-β-OH-His- Hin(139-190), a hybrid protein containing the DNA-binding domain of Hin recombinase and the putative iron-binding and oxygen-activating domain of the antitumor antibiotic bleomycin. This 54-residue protein, comprising residues 139-190 of Hin recombinase with the dipeptide pyrimidoblamic acid-β-hydroxy-L-histidine (PBA-β-OH-His) at the NH2 terminus, was synthesized by solid phase methods. PBA-β-OH-His-Hin(139- 190) binds specifically to DNA at four distinct Hin binding sites with affinities comparable to those of the unmodified Hin(139-190). In the presence of dithiothreitol (DTT), Fe•PB-β-OH-His-Hin(139-190) cleaves DNA with specificity remarkably similar to that of Fe•EDTA-Hin(139-190), although with lower efficiency. Analysis of the cleavage pattern suggests that DNA cleavage is mediated through a diffusible species, in contrast with cleavage by bleomycin, which occurs through a non-diffusible oxidant.

High and stable conversion efficiency obtaining in single-stage multi-crystal optical parametric chirped pulse amplification system

An optical parametric chirped-pulse amplification system is demonstrated to provide 32.9% pump-to-signal conversion efficiency . Special techniques are used to make the signal and pump pulses match with each other in both spectral and temporal domains. The broadband 9.5-mJ pulses are produced at the repetition rate of 1 Hz with the gain of over 1.9 x 10(8). The output energy fluctuation of 7.8% is achieved for the saturated amplification process against the pump fluctuation of 10%.