Biomedical subjects

M Eggert

Publications and source records attributed to M Eggert.

32 records · Page 2Linked to original sources

PubMed1995

A fraction enriched in a novel glucocorticoid receptor-interacting protein stimulates receptor-dependent transcription in vitro.

Glucocorticoids influence numerous cell functions by regulating gene activity. The glucocorticoid receptor (GR) is a ligand-activated transcription factor and, like any other transcription factor, does not modulate gene activity just by binding to DNA. Interaction with other proteins is probably required to enhance the establishment of a functional transcription initiation complex. To identify such proteins, we analyzed the in vitro interaction of the glucocorticoid receptor bound to a double glucocorticoid response element with nuclear proteins and describe here three interacting proteins with different molecular weights. One of them, which we named GRIP 170 (GR-interacting protein), was purified and microsequenced, and it turned out to be an unknown protein. When tested in a cell-free transcription assay, the fraction highly enriched for GRIP 170 does not influence basal promoter activity but does enhance GR induction.

Carrier Proteins↗

PubMed1995

Methylation of the mouse M-lysozyme downstream enhancer inhibits heterotetrameric GABP binding.

Expression of the mouse M-lysozyme gene is a specific marker for the differentiation of macrophage/granulocyte cell lineages. Analysis of the mechanisms regulating M-lysozyme gene expression revealed an enhancer element in the 3'-flanking region of the gene, termed the M-lysozyme downstream enhancer (MLDE). Here we demonstrate that the nuclear factors binding to MLDE are present in all tested myeloid and non-myeloid mouse cell lines. Sequence analysis of MLDE identified two different sequences, CAGGAAGT and CCGGAAGT, which match the consensus binding sequences for proteins of the ets gene superfamily. The two sites are oriented palindromicly and separated by 10 bp. DMS/DEPC interference assays revealed different patterns of DNA-protein contacts on the two sites. Mutation of each consensus sequence leads to an individual change in protein binding in vitro. Despite these differences, both sequences are bound by GABP, forming a heterotetrameric complex. Tissue specificity is correlated with demethylation of a single CpG dinucleotide located in one of the two Ets motifs. This site when methylated inhibits GABP binding to both sequences in non-macrophage cell types.

Animals↗

PubMed1993

Identification of novel phosphorylation sites in murine A-type lamins.

We report the distribution of phosphorylation sites in murine lamins A and C (A-type lamins) in vitro and in vivo followed by reverse-phase high-performance liquid chromatography and microsequencing of peptides spanning the almost complete lamin sequence. We show that two distinct protein kinases, cell-division-cycle-2 kinase (cdc2 kinase) and protein kinase C (PKC), phosphorylate murine A-type lamins at the non-alpha-helical amino- and carboxy-terminal domains in vitro and in vivo. Cdc2 kinase, but not PKC, is capable of inducing depolymerization of the nuclear lamina in permeabilized cells. Accordingly, lamins were proposed to be direct in vivo substrates of cdc2 kinase and PKC with different effects on the lamina dynamics. Analysis of the original A-type lamins revealed phosphorylation of residues Ser5 and Ser392. Residue Ser392 was substoichiometrically phosphorylated in the substrate and by cdc2 kinase in vitro. PKC phosphorylated peptides with its kinase-specific motifs surrounding Ser5, Thr199, Thr416, Thr480 and Ser625. In vivo, a mitosis-specific phosphorylation at the cdc2-kinase-specific phosphoacceptor site Ser392 and of the N-terminal peptide was identified. An interphase-specific phosphorylation at Ser525 matching the PKC consensus sequence and of peptides phosphorylated by unknown kinases was determined. The results lead us to propose that different cyclin-dependent kinase activities act as lamin kinases in mitosis and in interphase. Other kinases may cooperate with cdc2 kinase during reversible disassembly in mitosis and may modulate the supramolecular assembly of lamin filaments.

Amino Acid Sequence↗

PubMed1992

Parametric sequence comparisons.

Current algorithms can find optimal alignments of two nucleic acid or protein sequences, often by using dynamic programming. While the choice of algorithm penalty parameters greatly influences the quality of the resulting alignments, this choice has been done in an ad hoc manner. In this work, we present an algorithm to efficiently find the optimal alignments for all choices of the penalty parameters. It is then possible to systematically explore these alignments for those with the most biological or statistical interest. Several examples illustrate the method.

Algorithms↗

PubMed1991

Identification of phosphorylation sites on murine nuclear lamin C by RP-HPLC and microsequencing.

Isolated interphase lamin C, obtained from Ehrlich ascites tumor cells, was digested by Lys-C endoproteinase, the resulting peptides separated by reversed-phase HPLC and subjected to microsequencing in order to identify phosphorylation sites in interphase and following phosphorylation in vitro by cdc2-kinase, protein kinase C (PKC) and protein kinase A (PKA), respectively. Nuclear lamin C showed partial phosphorylation of Ser392 and Ser409, and possibly Ser407 in interphase. Phosphorylation was increased in response to cdc2-kinase at Ser390 and Ser392 and to PKC at Ser572. The N-terminal peptide (aa 1-32) containing consensus sequences for the 3 kinases was phosphorylated by cdc2-kinase, PKC and PKA. The sequence data suggests that multiple molecular switches via lamina modification control the dynamic behaviour of the nucleoskeleton during the cell cycle.

Amino Acid Sequence↗

PubMed1987

A new algorithm for best subsequence alignments with application to tRNA-rRNA comparisons.

The algorithm of Smith & Waterman for identification of maximally similar subsequences is extended to allow identification of all non-intersecting similar subsequences with similarity score at or above some preset level. The resulting alignments are found in order of score, with the highest scoring alignment first. In the case of single gaps or multiple gaps weighted linear with gap length, the algorithm is extremely efficient, taking very little time beyond that of the initial calculation of the matrix. The algorithm is applied to comparisons of tRNA-rRNA sequences from Escherichia coli. A statistical analysis is important for proper evaluation of the results, which differ substantially from the results of an earlier analysis of the same sequences by Bloch and colleagues.

Algorithms↗

PubMed1985

Rigorous pattern-recognition methods for DNA sequences. Analysis of promoter sequences from Escherichia coli.

The basic nature of the sequence features that define a promoter sequence for Escherichia coli RNA polymerase have been established by a variety of biochemical and genetic methods. We have developed rigorous analytical methods for finding unknown patterns that occur imperfectly in a set of several sequences, and have used them to examine a set of bacterial promoters. The algorithm easily discovers the "consensus" sequences for the -10 and -35 regions, which are essentially identical to the results of previous analyses, but requires no prior assumptions about the common patterns. By explicitly specifying the nature of the search for consensus sequences, we give a rigorous definition to this concept that should be widely applicable. We also have provided estimates for the statistical significance of common patterns discovered in sets of sequences. In addition to providing a rigorous basis for defining known consensus regions, we have found additional features in these promoters that may have functional significance. These added features were located on either side of the -35 region. The pattern 5', or upstream, from the -35 region was found using the standard alphabet (A, G, C and T), but the pattern between the -10 and the -35 regions was detectable only in a sub-alphabet. Recent results relating DNA sequence to helix conformation suggest that the former (upstream) pattern may have a functional significance. Possible roles in promoter function are discussed in this light, and an observation of altered promoter function involving the upstream region is reported that appears to support the suggestion of function in at least one case.

Base Sequence↗

PubMed1983

[Tryptophan content of cereal proteins].

The tryptophan contents of the albumin, globulin, prolamine and glutelin fractions from wheat, rye, barley, oat, sorghum, rice and maize were estimated by HPLC after alkaline hydrolysis. Complete amino acid compositions, including the amide contents, are given for all these protein fractions.

Amino Acids↗

PubMed1967