Biomedical subjects
Publications and source records attributed to M Bramucci.
The peptide isolated by us from calf pineal gland causes a reduction of RNA synthesis in vitro in L1210 and HL60 tumoral cells. This peptide also causes inhibition of cell proliferation; the cell viability is not modified. The effects are dose-dependent and reversible.
A variety of evidence suggests that a family of chromatin peptides (CPs), characterized by 1000D molecular weight, a pH dependent association to DNA and a prevailing presence of acidic amino acids in their structure, is involved in the regulation of genes expression. Nevertheless their action mechanism is still unknown. In our in vitro specific RNA transcription systems the CPs affect the initiation and not the elongation. Furthermore they inhibit the RNA transcription by interaction with the DNA rather than with the enzyme. The phagic in vitro specific RNA transcription is less affected by CPs than the eubacteric system, suggesting a kind of selectivity for target DNA sequences involved in the initiation of transcription.
RNA synthesis in cell and cell-free systems is inhibited by a family of acidic, low molecular weight chromatin peptides (CPs). These peptides were extracted from deproteinized DNA of prokaryotic and eukaryotic cells, but the low yield of purified material by this procedure hinders efforts aimed at understanding their action mechanism in gene regulation. In this report we describe two purification methods of CPs from an easily available source, wheat germ. A comparison is made between the method starting from deproteinized DNA and the method from purified chromatin. The biological effects (inhibition of L1210 cell growth and DNA in vitro transcription) of CPs from wheat germ together with their chemical characteristics (molecular weight, amino acid composition and presence of phosphoserine) show strong homology with those of CPs from other sources. These results suggest a possible role of these chromatin peptides in controlling gene expression.
The pentapeptide pyroGlu-Ala-Glu-Ser-Asn has been synthetized and phosphorylated in vitro at level of serine by protein kinase NII isolated from calf thymus chromatin. It is noteworthy that the calf thymus kinase NII shows a remarkable affinity for this peptide. The [32P]peptide is able to bind to several DNAs in the presence of Mg2+ (lambda phage, calf thymus, pBR540 plasmid). This binding appears not specific with regard to the type of DNA and its base sequence. These data support the hypothesis that phosphorylated acidic domains of nuclear nonhistone proteins could bind directly to DNA in the presence of Mg2+ cations.
Low molecular weight peptides have been isolated by alkali extraction from deproteinized DNA of E. coli cells grown in the presence of radioactive glutamic acid or orthophosphate. The labeled peptides, purified by gel filtration chromatography on Sephadex G25 and G10, contain prevailingly glutamic acid, aspartic acid, glycine, serine and alanine. Electrophoretic studies at different pH show that some peptide fractions contain a phosphoric residue. The N-terminus of the phosphorylated peptides is apparently blocked and they were able to bind to DNA in the presence of Mg2+ ions. Moreover the acidic peptides extracted from E. coli DNA show a sharp activity in the control of lambda phage DNA transcription 'in vitro'.
In the course of screening cDNA expression libraries with a monospecific polyclonal antibody to topoisomerase I, we isolated three different immunopositive cDNA clones. By comparing their derived amino acid sequences, a consensus region of similarity in otherwise completely dissimilar sequences was identified as an epitope. The approach described here should identify both continuous and discontinuous topological epitopes. The probability of occurrence of "spurious" immunopositive clones is shown to depend on the number of codons of each critical amino acid within the antigenic determinant.
Minimum substrate requirements for nuclear NII kinase (casein II kinase) were analyzed with synthetic peptides modeled according to amino acid composition of phosphopeptides isolated from chromatin. Uncharged blocked peptide termini decreased the requirement for acidic clusters neighboring the phosphate acceptor amino acid (serine) such that only one group immediately N-terminal to serine was sufficient for kinase activity. Studies on peptide interaction with DNA showed that the model phosphopeptides bound to DNA only in the phosphorylated form suggesting involvement of phosphorylation in protein-DNA interactions yet to be identified.
The partial amino acid sequence of rat topoisomerase I was determined by gas-phase microsequencing. Seven tryptic peptides closely matched the sequences deduced from human topoisomerase I cDNA (94.5% homology). Similarity to sequences deduced from baker's yeast and fission yeast genomic DNA were restricted to conserved domains which may represent important sites of interaction with DNA or with other proteins.
Low molecular weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction. They show very high specific activity in the control of transcription "in vitro". In this work the biochemical properties of controlling transcription peptide effectors isolated from trout testis DNA are reported. The purified peptides prevailingly contain glutamic acid, serine, aspartic acid, glycine and alanine. Studies of the peptide structure by N-terminal analysis using the dansyl chloride procedure was unsuccessful, suggesting the presence of a blocked NH2 group. At the same time the active peptides cannot be digested by carboxypeptidases. The gel filtration of the chromatin peptidic fractions on Sephadex G-25, Trisacryl GF05 or Sephadex G-15 shows that the active peptides elute as a single major peak with an elution volume corresponding to a molecular weight of about 1000. The paper electrophoresis performed at different pH and ionic strength shows that the chromatin peptides are separated in two fractions. One of them is strongly acidic and migrates towards the positive pole until pH 1.9, indicating the presence of phosphoric residues which probably exert an important role in the control of transcription "in vitro". The chromatin peptides are further purified by Sephadex G-10 and high performance liquid chromatography. The amino acid analysis of the purified peptides are reported.
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