PubMed Health⌕ Search

Biomedical subjects

M Czupryn

Publications and source records attributed to M Czupryn.

9 recordsLinked to original sources
PubMed1995

Alanine-scanning mutagenesis of human interleukin-11: identification of regions important for biological activity.

We have identified functionally important regions of human interleukin-11 (hIL-11) by means of alanine-scanning mutagenesis. A total of 61 mutated forms of hIL-11 were produced in E. coli as thioredoxin fusion proteins and tested in a murine T10 plasmacytoma proliferation assay. Mutations made at several positions proximal to the hIL-11 C-terminus caused substantial reduction in biological activity. In addition a number of other mutations in this region affected either protein folding or stability. Both effects displayed a characteristic periodicity with respect to the primary sequence which suggested that residues close to the C-terminus of hIL-11 adopt a helical conformation. Mutations made proximal to the N-terminus of hIL-11 also exhibited reduced bioactivity, although no effects on protein folding or stability were observed. The N-terminal mutations with reduced activity also mapped with a periodicity suggestive of a helical conformation. We previously have proposed a four-helix bundle topology for the hIL-11 structure based on physical studies, selective chemical modifications, positions of intron/exon boundaries, limit proteolysis experiments and site-directed mutagenesis. The alanine-scanning mutagenesis data we report here provide additional support for this model.

Alanine↗
PubMed1993

A Euglena gracilis zinc endonuclease.

A 26-kDa endonuclease has been purified to homogeneity from zinc-sufficient Euglena gracilis. The protein binds to single-stranded DNA with a higher affinity than to double-stranded DNA, but it exhibits nucleolytic activity toward both. Thus, it converts supercoiled plasmid pBR322 DNA into the linear form, a property characteristic of endonucleases, and it continues to act on the linearized DNA until it is completely degraded. It also hydrolyzes heat-denatured, single-stranded calf thymus DNA. Moreover, at amounts below 1 microgram, it enhances RNA synthesis by RNA polymerase II, a characteristic observed with other DNases. Its addition to an in vitro transcription assay increases RNA synthesis up to 3-fold. The nuclease requires two metal components to carry out its enzymatic activities. It hydrolyzes DNA only in the presence of millimolar amounts of magnesium or micromolar quantities of other activating metal ions, such as manganese, zinc, or cobalt. However, even when optimal concentrations of Mg2+ are present, micromolar amounts of the metal-chelating agents OP and HQSA completely inhibit pBR322 digestion. Transcription enhancement is also inhibited completely by both chelators at concentrations that do not affect the intrinsic polymerase II activity. By atomic absorption spectrometry, the enzyme contains 1 g-atom of Zn/mol, which is the likely target of chelator action. The nuclease protein can also be isolated from zinc-deficient E. gracilis, but remarkably it then contains 1 mol of Cu/g-atom and no zinc.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1992

Zinc rapidly induces a metal response element-binding factor.

Metal activation of metallothionein gene transcription is mediated by specific promoter sequences, termed metal regulatory elements (MREs). Nuclear extracts prepared from various human cell lines were assayed for their capacity to bind to a synthetic human MREa (hMREa) oligomer. Electrophoretic mobility-shift assays with extracts from control cells detected a single hMREa-containing complex. Addition to the growth medium of zinc, cadmium, or copper--metals known to induce MT biosynthesis in vivo--resulted in the rapid but reversible appearance of a second distinct hMREa-protein complex in all cell lines studied. This result was not seen when the metals were added directly to the extracts from control cells. DNA-binding protein blotting, UV crosslinking, and electroelution experiments were used to characterize the two hMREa-binding factors, termed BF1 and BF2. MRE-BF1 has an apparent molecular mass of approximately 86 kDa and binds to the hMREa in control cells, whereas MRE-BF2 consists of two molecules of approximately 28 kDa and binds to the hMREa in metal-treated cells. EDTA and o-phenanthroline inhibited binding of both factors to hMREa in a dose-dependent manner, indicating that a metal atom or atoms are essential for interaction of the factors with DNA.

Base Sequence↗
PubMed1988

Transcriptionally active chromatin can be selectively released by DNase I from Physarum polycephalum genome.

In a simple eukaryote Physarum polycephalum about 13% of the genome is transcribed into abundant cytoplasmic RNA as shown by S1 nuclease digestion of DNA-RNA hybrids. Mild digestion of isolated Physarum nuclei with DNase I liberates a fraction of chromatin 3.5-fold enriched in sequences hybridizing by Physarum poly(A)+ RNA. This fraction is similarly enriched in histone H4 and actin genes known to be actively transcribed in Physarum. High content (about 45%) of actively transcribed sequences in DNase-I-released fraction of Physarum chromatin makes it particularly well suited for studying the structural basis of transcriptional activation in eukaryotes.

Actins↗
PubMed1985

Lack of nucleosomal structure in a DNase-I-solubilized transcriptionally active chromatin fraction of Physarum polycephalum.

Light treatment of nuclei of Physarum polycephalum microplasmodia with DNase I, at low MgCl2 concentration (less than or equal to 3% DNA acid solubility, 0.1 mM MgCl2) selectively solubilizes a defined fraction of chromatin, in the form of a macromolecular complex. This fraction (up to 15% of the total chromatin) contains a full complement of the core histones and a reduced amount of histone H1, and is enriched in the high-mobility-group type of proteins. It is preferentially associated with nascent RNA and RNA polymerase B actively engaged in transcription. Digestion of DNAase-I-solubilized chromatin by micrococcal nuclease releases a size-heterogeneous population of cleavage products, indicative of lack of a typical nucleosomal packaging. It is concluded that the procedure used allows the isolation of structurally and functionally distinct regions of Physarum chromatin.

Chemical Phenomena↗
PubMed1980

Isolation and susceptibility to nucleases of transcriptionally active and inactive chromatin fractions from Physarum polycephalum.

Transcriptionally active and inactive chromatin fractions were isolated from Physarum polycephalum after depolymerization of chromatin with DNAase II or micrococcal nuclease, followed by fractionation in 5 mM-MgCl2. The active fraction of chromatin comprised up to 21% of nuclear DNA and was enriched 22-fold in the labelled nascent RNA. Both chromatin fractions were shown to have the nucleosomal structure. DNA of the active fraction of chromatin was degraded much faster with DNAase I and micrococcal nuclease than the DNA of the inactive fraction.

Chromatin↗