ABSTRACT
A prolyl endopeptidase was purified from Flavobacterium meningosepticum. It was digested with trypsin. Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment. A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands. It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids. Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da. The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase. In order to identify the active-site serine, the prolyl endopeptidase was labeled with [3H]diisopropyl fluorophosphate. One labeled peptide was purified and sequenced. The active-site serine was located in position 536 within the sequence GRSNGG. This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families.
Subject(s)
Endopeptidases/genetics , Flavobacterium/enzymology , Serine Endopeptidases , Amino Acid Sequence , Base Sequence , Binding Sites , Cloning, Molecular , DNA, Bacterial/genetics , Electrophoresis, Polyacrylamide Gel , Endopeptidases/metabolism , Isoflurophate/chemistry , Mass Spectrometry , Molecular Sequence Data , Nucleic Acid Hybridization , Open Reading Frames , Polymerase Chain Reaction , Prolyl Oligopeptidases , Protein Sorting Signals/isolation & purification , Restriction Mapping , Sequence Alignment , Trypsin/metabolismABSTRACT
We have examined the regulation of the synthesis of histone H1(0) in cultured mammalian cells treated with butyric acid. Treatment of cells with the inducer results in the arrest of synthesis of DNA and the other histones, while increasing the synthesis of H1(0) by a factor of 11. The induction of H1(0) by butyric acid occurs in a pulse with a peak at six hours, followed by a decrease to negligible levels. This pulse-like induction appears to be due to the fact that the cells are inducible for H1(0) only in the late S or G2 phases of the cell cycle. This, coupled with the fact that butyric acid blocks cells in G1, results in the burst of H1(0) synthesis after addition of the inducer. The G1 block provoked by butyric acid does not appear to result from the accumulation of H1(0). Removal of butyric acid from G1-blocked cells resulted in the resumption of cellular proliferation without prior loss of H1(0), demonstrating that the presence of this histone is not sufficient to prevent cellular proliferation.
Subject(s)
Butyrates/pharmacology , Cell Cycle/drug effects , Histones/biosynthesis , Animals , Butyric Acid , Culture Techniques , DNA/biosynthesis , Flow Cytometry , Interphase/drug effects , Kinetics , MiceABSTRACT
Histone H1(0) has a number of unusual properties that set it apart from other H1 subtypes (for review see ref. 1). For example, H1(0) synthesis is not strictly coupled to DNA synthesis, it is absent from the embryonic liver of mice (but present shortly after birth) and its synthesis is hormone-dependent in some of the glands of adult rodents. All the H1 subtypes differ in their DNA binding properties, and H1(0) has been shown to be preferentially associated with nuclease-resistant chromatin. These features suggest that the H1(0) may have a role in developmental gene control. To investigate this further, we have fractionated the H1(0)-containing nucleosomes of chromatin from adult mouse liver. We report here that the gene for alpha-fetoprotein, which is expressed in embryonic tissue but repressed soon after birth, is preferentially associated with the H1(0)-containing nucleosomes. The related gene for albumin, which is expressed in both embryonic and adult tissues, is absent from the H1(0)-containing nucleosome fraction. These results support a role for histone H1(0) in the control of gene expression.
Subject(s)
Gene Expression Regulation , Histones/genetics , Liver/metabolism , alpha-Fetoproteins/genetics , Albumins/genetics , Animals , Genes , Genes, Developmental , MiceABSTRACT
This report describes experiments designed to study the organization of the linker DNA in nucleosomes. When rat liver nucleosomes (145 to 188 base pairs in length) were digested by Exonuclease III and then by nuclease S1 a series of bands on sizes 90 - 102 - 112 - 125 - 135 - 142 - 154 - 166 - 172 - 181 - bases was observed in denaturing electrophoretic gels. Digestion of H1-depleted nucleosomes under the same conditions results in a series of products of sizes (10.4) xn in base (n less than or equal to 14) only. This result is interpreted as reflecting a particular arrangement of linker DNA under the influence of histone H1.
Subject(s)
DNA/physiology , Exodeoxyribonucleases , Histones , Animals , Base Composition , Chemical Phenomena , Chemistry , Deoxyribonucleases , Endonucleases , Exonucleases , Liver , Nucleic Acid Denaturation , Nucleosomes , Rats , Single-Strand Specific DNA and RNA EndonucleasesABSTRACT
The mode of interaction of histone H1 with the nucleosome is governed by the relative distribution of the linker with respect to the core DNA. Preliminary experiments (Simpson, R.T. 1978, Biochemistry, 17, 5524-5531) and tentative models (Thoma, F. et al. (1979), J. Cell. Biol., 83, 403-427) suggest that part of the linker complete two full turns of DNA around the histone core, probably by adding 10 base pairs at each end of the core DNA. In the present study Exonuclease III has been utilized to digest the 3' ends of H1 depleted nucleosomes. (i.e. the 195 base pair particle). The analysis of the resulting DNA fragments under denaturing conditions shows that the whole linker is distributed symmetrically with respect to the core DNA.
