ABSTRACT
LINEs are transposable elements, widely distributed among eukaryotes, that move via reverse transcription of an RNA intermediate. Mammalian LINEs have two ORFs (ORF1 and ORF2). The proteins encoded by these ORFs play important roles in the retrotransposition process. Although the predicted amino acid sequence of ORF1 is not closely related to any known proteins, it is highly basic; thus, it has long been hypothesized that ORF1 protein functions to bind LINE-1 (L1) RNA during retrotransposition. Cofractionation of ORF1 protein and L1 RNA in extracts from both mouse and human embryonal carcinoma cells indicated that ORF1 protein binds L1 RNA, forming a ribonucleoprotein particle. Based on UV crosslinking and electrophoretic mobility-shift assays using purified components, we demonstrate here that the ORF1 protein encoded by mouse L1 binds nucleic acids with a strong preference for RNA and other single-stranded nucleic acids. Furthermore, multiple copies of ORF1 protein appear to bind single-stranded nucleic acid in a manner suggesting positive cooperativity; such binding characteristics are likely to be facilitated by the protein-protein interactions detected among molecules of ORF1 polypeptide by coimmunoprecipitation. These observations are consistent with the formation of ribonucleoprotein particles containing L1 RNA and ORF1 protein and provide additional evidence for the role of ORF1 protein during retrotransposition of L1.
Subject(s)
Open Reading Frames , Retroelements , Ribonucleoproteins/genetics , Animals , Humans , Mice , Plasmids , Protein Binding , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
The mouse LINE-1 (L1) retrotransposon contains two open reading frames (ORFs). Three classes of the protein encoded by the first open reading frame (ORF1) are expressed in the mouse embryonal carcinoma cell line, F9; the apparent molecular sizes of these proteins are 41.3, 43, and 43.5 kDa. Two of these three proteins (41.3 and 43 kDa) are translated in vitro from full-length, sense-strand L1 RNA isolated from ribonucleoprotein particles. A reverse transcription-polymerase chain reaction approach was used to clone the ORF1 region from RNA isolated from ribonucleoprotein particles, then the coding capacity of these clones was examined using in vitro transcription and translation. Multiple sequences that encode ORF1 were recovered by this approach, indicating that multiple loci of L1 in the mouse genome are expressed in F9 cells. In addition, L1 sequences with intact ORF1 regions appear to be selectively enriched in the ribonucleoprotein particles.
Subject(s)
Polymorphism, Genetic , Retroelements , Ribonucleoproteins/genetics , Animals , Base Sequence , Cloning, Molecular , DNA Primers , Mice , Molecular Sequence Data , Open Reading Frames , Polymerase Chain Reaction , Protein Biosynthesis , Tumor Cells, CulturedABSTRACT
The complete nucleotide sequence of Citrus limon 26S rDNA has been determined. The sequence has been aligned with large ribosomal RNA (L-rRNA) sequences of Escherichia coli, Saccharomyces cerevisiae and Oryza sativa. Nine extensive expansion segments in dicot 26S rRNA relative to E. coli 23S rRNA have been identified and compared with analogous segments of monocot, yeast, amphibian and human L-rRNAs. A secondary structure model for lemon 26S rRNA has been derived based on the refined model of E. coli 23S rRNA. It has been compared with other eukaryotic L-rRNAs models in terms of location of functionally important regions. Origin and evolution of L-rRNA expansion segments are discussed.
Subject(s)
DNA, Ribosomal/genetics , Plants/genetics , RNA, Ribosomal/genetics , Base Sequence , Escherichia coli/genetics , Hydrogen Bonding , Models, Structural , Molecular Sequence Data , Nucleic Acid Conformation , Plasmids , RNA, Ribosomal, 23S/genetics , Sequence Homology, Nucleic AcidABSTRACT
Specific endonuclease activities have been found it two Pseudomonas aeruginosa strains. Isolation and purification of enzymes and determining their specific activities have permitted one to find out that PaeI is an isoshizomer of SphI and digests the sequence 5'-GCATG C-3'. Another isolated enzyme PaeII is an isoshizomer of SmaI and cleaves DNA in a fragment 5'-CCC GGG-3'. The use of PaeI and PaeII enzymes in genetical engineering and their advantages are discussed.
Subject(s)
DNA Restriction Enzymes/analysis , Deoxyribonucleases, Type II Site-Specific , Pseudomonas aeruginosa/enzymology , Base Sequence , DNA Restriction Enzymes/genetics , Electrophoresis, Polyacrylamide Gel , Genetic Engineering , Pseudomonas aeruginosa/geneticsABSTRACT
PaeI, a new restriction endonuclease from Pseudomonas aeruginosa clinical strain was isolated and characterized. It recognizes and cleaves the sequence 5'-GCATG reduced C-3' generating DNA fragments with 3'-tetranucleotide sticky ends. DNAs of pBR322, SV40 and bacteriophage lambda have one, two and six PaeI recognition sites, respectively. Seventy-two strains of Pseudomonas, Clostridium, Escherichia coli, Shigella, Proteus and Saccharomyces were screened for the presence of site-specific endonucleases. Here we describe the PaeI restriction enzyme found in Pseudomonas aeruginosa; other data will be published elsewhere. Earlier Hinkle and Miller isolated from P. aeruginosa a PaeR7 restriction endonuclease recognizing and cleaving a sequence 5'-C reduced TCGAG-3' (1). Sequence analysis of DNAs cleaved by PaeI shows that the enzyme is the isoschizomer of SphI (2).
