ABSTRACT
Many genes, particularly those encoding the products participating in the regulation of transcription, replication and tissue remodeling, produce short-lived mRNA. It has been commonly accepted that once mRNA is disintegrated, the degradation process is so rapid that the decay intermediates cannot be detected. In the present study we verified this postulate and focused our attention on the quantification of the decay products of the urokinase-type plasminogen activator (uPA) mRNA that belongs to short-lived mRNAs. Using a previously described modified quantitative RT-PCR method, we have shown that intact uPA mRNA coexists in normal human tissues, Jurkat and 5637 cells with a great abundance of its degradation products. The uPA mRNA decay products were not detected in T24P cells. The content of intact uPA mRNA in normal tissues was as low as 5% of the total amount of its poly(A)(+) fraction. The size distribution of the mRNA decay products suggests that the mRNA is digested by exonucleases or/and non-specific endonuclease with cut sites evenly distributed along the mRNA chain. Different decay degrees were demonstrated for subpopulation of the uPA mRNA molecules with intact 3' and 5' ends.
Subject(s)
RNA, Messenger/chemistry , Urokinase-Type Plasminogen Activator/genetics , Blotting, Northern , Cell Line , DNA, Complementary/chemistry , Enzyme Stability , Humans , Kidney/enzymology , Lung/enzymology , Particle Size , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Tumor Cells, Cultured , Urokinase-Type Plasminogen Activator/chemistryABSTRACT
52 strains of Bacillus generum have been tested for production of site-specific endonucleases. The sequence recognized by the enzyme was determined for 23 enzymes, the cleavage site inside the sequence was determined for 5 enzymes. All the enzymes under study were found to be isomers of the known enzymes. The selected strains are peculiar for the high level of site-specific endonucleases content and may be used as producents of the enzymes.
Subject(s)
Bacillus/enzymology , DNA Restriction Enzymes/isolation & purification , Substrate SpecificityABSTRACT
The sitespecific restriction endonucleases were found in four strains among the twelve strains of anaerobic bacteria of generum Bifidobacterium. Two of the restriction endonucleases studied, BadI from B. adolescentis LVA1 and BbfI from B. bifidum LVA3, are isoshizomers of XhoI and recognize the nucleotide sequence CTCGAG. The restriction endonucleases Bbf7411I from B. bifidum 7411 and Bla7920I from B. lactentis 7920 recognize and hydrolize the nucleotide sequence TCCGGA having the specifity analogous to the one of restriction endonuclease CauB3I. Like CauB3I, these restriction endonucleases are unable to hydrolyize DNA if the adenine residues in the recognition site are methylated.
