Multicopy single-stranded DNA isolated from a gram-negative bacterium, Myxococcus xanthus

A gram-negative bacterium, Myxococcus xanthus, was found to contain 500 to 700 copies per chromosome of a short single-stranded linear DNA fragment. When this DNA (multicopy single-stranded DNA; msDNA) labeled at the 5' end with kinase was used as a probe against total chromosomal blots, it hybridized to unique high molecular weight bands, which were cloned and sequenced. Labeling of msDNA was also possible using the Klenow fragment of DNA polymerase I as well as terminal deoxynucleotidyl transferase, permitting direct sequencing. The 5' end of msDNA was found to be primed by a short RNA segment. The DNA portion of msDNA consisted of 163 bases. Exact correspondence was seen between the msDNA sequence and the sequence of a chromosomal clone. An elaborate secondary structure is postulated for the msDNA sequence. A similar satellite DNA was also found in another myxobacterium, Stigmatella aurantiaca.     

Distribution of multicopy single-stranded DNA among myxobacteria and related species.

Multicopy single-stranded DNA (msDNA) is a short single-stranded linear DNA originally discovered in Myxococcus xanthus and subsequently found in Stigmatella aurantiaca. It exists at an estimated 500 to 700 copies per chromosome (T. Yee, T. Furuichi, S. Inouye, and M. Inouye, Cell 38:203-209, 1984). We found msDNA in other myxobacteria, including Myxococcus coralloides, Cystobacter violaceus, Cystobacter ferrugineus (Cbfe17), Nannocystis exedens, and nine independently isolated strains of M. xanthus. The presence of msDNA in N. exedens would extend its phylogenetic distribution into another family of myxobacteria. Flexibacter elegans, a Cytophaga-like gliding bacteria which may be even more distantly related, also contained an msDNA but at a much lower copy number. msDNA was not detected in closely related strains of the myxobacteria Cystobacter fuscus and C. ferrugineus (Cbfe16 and Cbfe18) and the more distantly related eubacteria Herpetosiphon giganteus, Taxeobacter ocellatus, Lysobacter antibioticus, Lysobacter enzymogenes, Cytophaga johnsonae, Rhodopseudomonas sphaeroides, and Rhodospirillum rubrum. Thus far, msDNA has been found in certain gliding bacteria but not in others.     

Biosynthesis and structure of stable branched RNA covalently linked to the 5' end of multicopy single-stranded DNA of Stigmatella aurantiaca

Stigmatella aurantiaca, a gram-negative bacterium, contains approximately 500 copies per cell of a short single-stranded linear DNA (multicopy single-stranded DNA: msDNA). This DNA is attached to a branched RNA (msdRNA) by its 5' end. The entire sequence of msdRNA was determined and found to consist of 76 bases. The msDNA is linked at the 19th G residue of msdRNA by a 2', 5' phosphodiester linkage. The coding region for msdRNA (msr) is located downstream of the coding region for msDNA (msd). These coding regions exist in opposite orientation with respect to each other and overlap by 8 bases at their 3' ends. Biosynthesis of RNA-linked msDNA was characterized and mechanisms of synthesis are proposed.     

Structure of msDNA from Myxococcus xanthus: evidence for a long, self-annealing RNA precursor for the covalently linked, branched RNA

The branched RNA (msdRNA) of M. xanthus consists of 77 bases. The 20th rG residue is linked to the 5' end of msDNA, consisting of 162 bases, by a 2', 5' phosphodiester linkage. The msdRNA coding region is located on the chromosome in the opposite orientation to the msDNA coding region, with the 3' ends overlapping by eight bases. S1 nuclease mapping experiments indicate that the primary product of msdRNA is much longer at both the 5' and 3' ends (approximately 375 bases). Because of homologous sequences upstream of the msdRNA and msDNA coding regions, the precursor RNA molecule is considered to form an extremely stable stem-and-loop structure (delta G = -210 kcal). We propose a novel mechanism of DNA synthesis in which the stem-and-loop structure serves as a primer as well as a template to form the branched RNA-linked msDNA.     

A mutational study of the site-specific cleavage of EC83, a multicopy single-stranded DNA (msDNA): nucleotides at the msDNA stem are important for its cleavage.

Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA. Such molecules are encoded by genetic elements called retrons. Unlike other retrons, retron EC83 isolated from Escherichia coli 161 produces RNA-free msDNA by site-specific cleavage of msDNA at 5'-TTGA/A-3', where the slash indicates the cleavage site. In order to investigate factors responsible for the msDNA cleavage, retron EC83 was treated with hydroxylamine and colonies were screened for cleavage-negative mutants. We isolated three mutants which were defective in msDNA cleavage and produced RNA-linked msDNA. They were all affected in msd, a gene for msDNA, with a base substitution at the bottom part of the msDNA stem. In contrast, base substitution at and around the cleavage site has no marked effect on msDNA synthesis or its cleavage. From these results, we concluded that the nucleotides at the bottom of the msDNA stem, but not the nucleotides at the cleavage site, play a major role in the recognition and cleavage of msDNA EC83.     

A general strategy for cloning double-stranded RNA: nucleotide sequence of the Simian-11 rotavirus gene 8

Using Simian-11 rotavirus RNA, a strategy has been developed for the production of full length cloned copies of the genes of double-stranded (dsRNA) viruses. Genomic RNA segments were polyadenylated and reverse transcribed to yield a mixture of full length cDNA copies of both possible polarities. The cDNAs were annealed, filled in to complete any partial copies, tailed and inserted into the PstI site of pBR322 using dG/dC tailing. Cloned rotavirus cDNA gene copies were assigned to genomic RNA segments by Northern hybridization. The complete sequence of gene 8 which codes for NCVP3, a non-structural protein of SA11 rotavirus, was determined from a cloned gene copy. It is 1059 bases in length and has an open reading frame which could code for a protein containing 317 amino acids. The apparent 5' and 3' terminal non coding regions are 46 and 59 bases in length, respectively. The sequence ATGTGACCOH at the 3' end of the plus strand is conserved in four of the eleven genes examined. The cloning procedures used should be generally applicable to viruses with segmented dsRNA genomes.     

Branched RNA covalently linked to the 5' end of a single-stranded DNA in Stigmatella aurantiaca: structure of msDNA

Stigmatella aurantiaca is a gliding, gram-negative bacterium that shows a spectacular fruiting body formation upon starvation of nutrient. This bacterium was found to contain approximately 500 copies per cell of a short single-stranded linear DNA (multicopy single-stranded DNA: msDNA). The primary structure of msDNA was determined and found to consist of 162 or 163 deoxyribonucleotides. Its unique chromosomal gene was cloned and sequenced. The msDNA was found to be attached to a branched RNA by its 5' end. Structural analysis of the branched RNA revealed that it consists of a triribonucleotide, 5'A-G-(C or U)3', and that msDNA is branched out from the 2' position of the rG residue forming a 2', 5' phosphodiester linkage with the dC residue at the 5' end of msDNA.     

Structural requirements of the RNA precursor for the biosynthesis of the branched RNA-linked multicopy single-stranded DNA of Myxococcus xanthus

A precursor RNA molecule (pre-msdRNA) of approximately 375 bases is considered to form a stable secondary structure which serves as a primer as well as a template to synthesize the branched RNA-linked multicopy single-stranded DNA (msDNA) of Myxococcus xanthus. When 3-base mismatches were introduced into the stem structure immediately upstream of the branched rG residue to which msDNA is linked by a 2',5'-phosphodiester linkage, the production of msDNA was almost completely blocked. However, if additional 3-base substitutions were made on the other strand to resume the complementary base pairing, msDNA production was restored, being consistent with the proposed model of msDNA synthesis. We also found that the branched rG residue of pre-msdRNA could not be replaced with either rC or rA, while the 5' end (dC) of msDNA which is linked to the branched rG could be substituted with a dG residue. Together with several other mutations, the structural requirements of pre-msdRNA are discussed with respect to the mechanism of msDNA biosynthesis.