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Biomedical subjects

K Luger

Publications and source records attributed to K Luger.

25 records · Page 2Linked to original sources
PubMed1996

Molecular cloning and expression of mouse procalcitonin.

The nucleotide sequence for mouse calcitonin was determined from a cDNA obtained using a polymerase chain reaction (PCR) based method, the rapid amplification of cDNA ends (RACE)-PCR. Primers designed from highly conserved regions in the coding sequences of known rat and human calcitonin cDNAs were used to amplify calcitonin cDNA as 5'-end and 3'-end fragments from mouse thyroid RNA. The obtained cDNA is 850 bp in length most probably representing the entire mouse calcitonin mRNA. It contains an open reading frame coding for a 136 amino acid protein with a calculated M(r) of 15,143. Comparison of the deduced amino acid sequences of preprocalcitonin of mice with other species revealed highest homologies to the rat (93%) and human (77%) sequences. A recombinant form of mouse precalcitonin (rmPCT) of approximately 17 kDa was expressed as a fusion peptide in E.coli transformed with a PCR-cloned expression construct.

Amino Acid Sequence↗
PubMed1996

Mapping nucleosome position at single base-pair resolution by using site-directed hydroxyl radicals.

A base-pair resolution method for determining nucleosome position in vitro has been developed to com- plement existing, less accurate methods. Cysteaminyl EDTA was tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specifically by hydroxyl radical-catalyzed chain scission by using the Fenton reaction. Strand cleavage occurs mainly at a single nucleotide close to the dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA. The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegatus 5S rRNA sequence and a twofold-symmetric derivative. The weakness of translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demonstrated. The predominant location of both histone octamer and H3H4 tetramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucleosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptable for determining nucleosome position in chromatin in vivo.

Animals↗
PubMed1991

Autonomous folding and coenzyme binding of the excised pyridoxal 5'-phosphate binding domain of aspartate aminotransferase from Escherichia coli.

The coenzyme (PLP) binding domain (residues 47-329) of the dimeric aspartate aminotransferase from Escherichia coli was produced separately by recombinant DNA methods. It folded autonomously both in vivo and in vitro, that is, independently of the native N- and C-terminal extensions that combine to form the small domain of eAAT. The PLP-domain had one binding site for PLP of relatively high affinity involving a covalent bond to the protein. It was monomeric, although the major subunit-subunit interface at the 2-fold symmetry axis remained unchanged. This effect appears to be due mainly to the absence of the N-terminal extension that contains hydrophobic residues, which interact with the PLP-domain of the second subunit in the wild-type dimer. Judged by circular dichroism, fluorescence, and HPLC gel filtration at increasing concentrations of guanidinium chloride, the PLP-domain underwent a three-state unfolding transition (M' in equilibrium M'* in equilibrium U') involving a compact intermediate M'*. This behavior parallels the unfolding of the dissociated native monomer of cAAT.

Apoenzymes↗
PubMed1990

An 8-fold beta alpha barrel protein with redundant folding possibilities.

Protein sequences containing redundant segments of secondary structure at both termini have the choice a priori of folding into several possible circularly permuted variants of the wild-type tertiary structure. To test this hypothesis the gene of phosphoribosyl anthranilate isomerase from yeast, which is a single-domain 8-fold beta alpha barrel protein, was modified to produce a 10-fold beta alpha homologue in Escherichia coli. It contained a duplicate of the two C-terminal beta alpha units of supersecondary structure fused to its N-terminus. Most of the protein was recovered from the insoluble fraction of disrupted cells by dissolution in guanidinium chloride solutions and refolding. Pristine protein was purified from the soluble fraction. The purified (beta alpha)10 proteins were enzymically almost fully active. Absorbance, fluorescence and circular dichroism spectra as well as the reversible unfolding behaviour of both proteins were also very similar to the properties of the original (beta alpha)8 protein. Digestion with endopeptidases converted both the pristine and the refolded (beta alpha)10 variant to the same large fragment that had the N-terminal sequence and mol. wt of the wild-type (beta alpha)8 protein. The data suggest that the folding of the (beta alpha)10 variant is controlled thermodynamically both in vivo and in vitro.

Aldose-Ketose Isomerases↗
PubMed1989

Correct folding of circularly permuted variants of a beta alpha barrel enzyme in vivo.

An important question in protein folding is whether the natural amino and carboxyl termini and the given order of secondary structure segments are critical to the stability and to the folding pathway of proteins. Here it is shown that two circularly permuted versions of the gene of a single-domain beta alpha barrel enzyme can be expressed in Escherichia coli. The variants are enzymically active and are practically indistinguishable from the original enzyme by several structural and spectroscopic criteria, despite the creation of new termini and the cleavage of a surface loop. This novel genetic approach should be useful for protein folding studies both in vitro and in vivo.

Aldose-Ketose Isomerases↗
PubMed1988

The role of the TRP1 gene in yeast tryptophan biosynthesis.

Transcription of the gene for phosphoribosyl-anthranilate isomerase (TRP1) from the TRP1 promoter is initiated only approximately half as frequently as, for example, from the TRP3 promoter, but TRP1 mRNA is approximately twice as stable as TRP3 mRNA. Therefore, the steady state amount of TRP1 mRNA in yeast cells, grown without amino acid limitation, is similar to the steady-state amount of TRP3 mRNA. The protein concentration of both enzymes in yeast cells is about the same, but the basal specific enzyme activity in permeabilized cells of the TRP1 gene product N-(5'-phosphoribosyl-1)-anthranilate isomerase is about 2-3 times higher than that of any of the other TRP enzymes. According to the kinetic parameters of the purified isomerase protein, the enzyme is more active than, for example, the purified TRP3 enzyme indoleglycerol-phosphate synthase. It is suggested that the TRP1 gene of Saccharomyces cerevisiae might be the result of a rearrangement event, separating the N-(5'-phosphoribosyl-1)-anthranilate isomerase domain from the indoleglycerol-phosphate synthase domain and putting the catalytically more active isomerase domain behind a weak and nonregulated constitutive promoter.

Aldose-Ketose Isomerases↗