ABSTRACT
Analysis of the blood serum of healthy and infected with malaria plasmodium mice showed a steep rise in content of linear double-stranded DNA (0.2-0.5 and 2-15 gamma/ml, respectively). Some physico-chemical properties of serum DNA and a DNA-associated glycoprotein (M approximately 40 kDa) are determined.
Subject(s)
Malaria/blood , Nucleoproteins/blood , Amino Acids/analysis , Animals , DNA/chemistry , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Malaria/veterinary , Mice , Nucleoproteins/isolation & purificationABSTRACT
From the tryptic hydrolysis of 40-kDa protein from the mouse blood serum thirty-four peptides were isolated by HPLC, of which complete and partial amino acid sequence was established for twenty-eight and six, respectively. On the basis of these data the protein is identified as the blood serum alpha 1-acid glycoprotein.
Subject(s)
Blood Proteins/chemistry , Nucleoproteins/blood , Orosomucoid/chemistry , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Hydrolysis , Malaria/blood , Malaria/veterinary , Mice , Molecular Sequence Data , TrypsinABSTRACT
Interaction of MspI restriction endonuclease with a series of oligodeoxynucleotides, varying in stability of secondary structure and in location of the restriction site, has been studied. It is shown that a functionally active MspI-site must be double-stranded and flanked from both sides. Separate MspI-cleavage of dodecanucleotides dCGACCCGGGATC and dGATCCCGGGTCG is inhibited by the reaction products as well as by non-homological hexanucleotides dGGTACC and dGGATCC (but not by dCGGCGC). Polyethylene glycol in low concentrations (1-3%) promotes and in higher concentrations (7-14%) inhibits the cleavage. A scheme of MspI functioning is suggested including enzyme's step-by-step recognition of the restriction site and its nonspecific interaction with flanking segments of DNA, which leads to formation of the productive complex.
Subject(s)
DNA Restriction Enzymes/metabolism , Oligodeoxyribonucleotides/metabolism , Base Sequence , Binding Sites , Deoxyribonuclease HpaII , Electrophoresis, Polyacrylamide Gel , Nucleic Acid Heteroduplexes , Oligodeoxyribonucleotides/chemical synthesis , Protein Conformation , Substrate SpecificityABSTRACT
Hybridization of synthetic oligodeoxynucleotides with single-stranded phage M13mp2 DNA has been studied in terms of temperature, ionic strength, oligonucleotide molar excess and chain length, and DNA secondary structure. Combination of two decadeoxynucleotides corresponding to a nicked eicosamer (composite primer) was found to be efficient in the template-directed DNA polymerase-catalyzed chain elongation, where both decamers separately failed. Circular SS DNA was specifically linearized by BamHI cleavage of SS DNA-tetradecadeoxynucleotide duplex.
Subject(s)
DNA Restriction Enzymes , DNA, Single-Stranded/analysis , DNA-Directed DNA Polymerase , Nucleic Acid Conformation , Nucleic Acid Hybridization , Oligodeoxyribonucleotides/analysis , Coliphages/analysis , DNA, Viral/analysis , Electrophoresis, Polyacrylamide GelABSTRACT
Interaction of the restriction endonuclease BamHI with a series of synthetic oligodeoxynucleotides containing the restriction site has been studied. The enzyme is shown to specifically cut the BamHI site in hexanucleotide (I) and in non-selfcomplementary deca- and octanucleotides (II)-(IV). The data obtained led to the conclusion that BamHI reacts with duplex structures, while playing an important role in their stabilization. In 14-mer (V) BamHI cuts a non-standard half-site GAA to yield the 5'-terminal tri- (rather than hepta-) nucleotide. Hypothetical mechanisms of the process are discussed basing on conception of the role of higher DNA structures in the interaction with restriction endonucleases.
Subject(s)
DNA Restriction Enzymes/metabolism , Oligodeoxyribonucleotides/metabolism , Base Sequence , Deoxyribonuclease BamHI , Electrophoresis, Polyacrylamide Gel , In Vitro Techniques , Macromolecular Substances , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemical synthesisABSTRACT
In the course of studying extrachromosomal DNA with composite replicons, a hybrid has been constructed by the in vitro recombination of the filamentous phage M13mp2 DNA (RF) and plasmid pUR222 (ApR). Both parental DNAs contain a fragment of lac-operon (ca. 800 bp), which includes the distal end of lacI gene, lacPO segments, and the lacZ gene proximal region coding for 145 N-terminal amino acid residues of beta-galactosidase and thus providing for alpha-complementation, the effect being cancelled with a polynucleotide insertion at the unique EcoRI site in the lacZ gene segment. E. coli BMH71-18 cells were transformed with the ligated mixture of EcoRI restricts of both DNAs. A phage-like nucleoprotein was isolated from colourless plaques (on the Xgal- and IPTG-supplemented medium); its deproteinization yielded a DNA which contains the ApR-determinant and, according to PAGE, structurally specific staining, restriction analysis, sequencing by the Sanger procedure, and electron microscopy data, is a linear double-stranded molecule comprising the phage and plasmid genomes in an equimolar ratio. Since the hybrid DNA does not display the alpha-complementation effect, both bacterial inserts are in the opposite orientation. Transformation of both phage (F+) and plasmid (F-) hosts with the hybrid DNA led to cultures which, after precipitation of the nucleoprotein from the extracellular medium and deproteinization, afforded the same composite DNA.(ABSTRACT TRUNCATED AT 250 WORDS)