Previous work has demonstrated the presence of a self‐splicing intron in the large subunit ribosomal RNA coding region in some strains of the ciliate protozoan Tetrahymena. Sequence comparisons of the intron regions from six Tetrahymena species showed these to fall into three homology groups. In an attempt to evaluate the evolutionary origins of the intervening sequences, we have now determined complete small subunit ribosomal RNA gene sequences from 13 species of Tetrahymena and the absolute number of nucleotide differences between the sequences was used to construct a phylogenetic tree. This phylogeny was consistent with the groupings suggested by comparisons of other biochemical characters including cytoskeletal proteins, isozyme analyses, and restriction maps of complete rRNA transcription units. The homology groupings that were based upon the intron sequence data do not agree with the relationships inferred from the small subunit rRNA sequence data. These observations are taken to indicate that the intron character has been acquired independently in different species at a stage later than the branching out of the species.

The bsgA gene of Myxococcus xanthus plays an essential role in the regulation of early gene expression during fruiting body formation and sporulation. bsgA mutants behave as though unable to initiate a required cell-cell interaction and consequently fail to transcribe normal levels of many developmentally induced genes. We determined the nucleotide sequence of bsgA, which predicts a single gene encoding a 90.4-kDa protein. The deduced BsgA protein shares 45 and 48% amino acid identity with the lon genes of Escherichia coli and Bacillus brevis, respectively. The cloned bsgA gene was expressed in E. coli, and the BsgA protein was partially purified and found, like its E. coli homolog, to be an ATP-dependent protease. Thus, the basis for the phenotype of bsgA mutants is likely to be a defect in intracellular proteolysis.

The yeast mitochondrial RNA polymerase (RNAP) is a two-subunit enzyme composed of a catalytic core (Rpo41) and a specificity factor (Mtf1) encoded by nuclear genes. Neither subunit on its own interacts with promoter DNA, but the combined holo-RNAP recognizes and selectively initiates from promoters related to the consensus sequence ATATAAGTA. To pursue the question of why Rpo41, which resembles the single polypeptide RNAPs from bacteriophage T7 and T3, requires a separate specificity factor, we analyzed a collection of Mtf1 point mutations that confer an in vivo petite phenotype. These mutant proteins are able to interact with Rpo41 and are capable of nearly wild type levels of initiation in vitro with a consensus promoter-containing template (14 S rRNA). However, the petite phenotype of two mutants can be explained by the fact that they exhibit dramatic transcriptional defects on non-consensus promoters. Y54F is incapable of transcribing the weak tRNA Cys promoter, and C192F cannot transcribe either tRNA Cys or the variant COX2 promoter from linear DNA templates. Transcription of the tRNA Cys promoter by both mutants was significantly corrected by addition of an initiating dinucleotide primer or by supercoiling the DNA template. These results establish the critical role of Mtf1 in promoter recognition and initiation of transcription.

Amplification of bacteriophage Mu DNA following infection of a sensitive host or induction of a Mu lysogen is accomplished by a series of replicative transposition events (for reviews, see references 15 and 21). Host-encoded requirements for this process include the DNA replication proteins and proteins which affect DNA structure: IHF, HU, and gyrase. The phage-encoded requirements include the A (transposase) and B proteins, the termini of the Mu genome, which carry binding sites for the transposase (3), and an enhancer sequence located near the left terminus which is involved in correct alignment of the termini (10,19,29). In addition, an unusually strong DNA gyrase binding site (SGS), located near the center of the Mu genome, is required for efficient Mu replication (22,23). Deletion of 147 bp containing the gyrase binding site from a Mu prophage results in a delay of replication for about an hour after induction.The host DNA gyrase, along with topoisomerase I, is required for maintaining a proper degree of superhelicity in cellular DNA (for a review, see reference 4), and alterations in superhelicity have been implicated in the regulation of gene expression in a large number of biological systems (for a review, see reference 5). However, the existence of unusual gyrase binding sites, such as the Mu SGS and a site in pSC101 that is required for plasmid maintenance (32), indicates that gyrase may play a more specific role in some biological processes. Mu requires gyrase for replication (24,37), but what specific requirements gyrase fulfills and what role the SGS plays compared with numerous weaker sites in the Mu genome remain to be elucidated.There are several stages at which replication of prophage lacking the SGS could be inhibited. These include (i) early transcription and translation, which yield the required A and B proteins; (ii) synapsis of the prophage termini through oligomerization of transposase molecules bound at the termini (18); (iii) formation of a ''cleaved-donor complex'' (3) (also called type I transpososome [28]) by introduction of singlestrand cleavages at the Mu termini; (iv) formation of a ''strand transfer complex'' (16) (also called type II transpososome [28]), which includes synapsis with target DNA and strand transfer; and (v) binding of host replication machinery and replication itself (7). In this study we characterized Mu prophages with a deleted or altered SGS, and here we describe the effects of the changes on various stages of the replication pathway. MATERIALS AND METHODSBacteria, bacteriophage, and plasmids. Strain X1 is Escherichia coli K-12 AB1157 that is recB recC sbcB and monolysogenic for Mucts62. MP1594 is W3110 thy lacZ::Mucts62. MP507 is W3110 thy malB::Mu BAm1066. The phages used were Mucts62, Mu pAp1, and Mu nuB1 (37). Plasmid pBR322 was used for cloning of Mu DNA fragments. Cultures were grown in L broth, except for the experiments requiring thymine starvation, when T medium supplemented with 20 g of thymine per ml and 0.4% glucose was used (13).Prophage constructio...

HeLa and 293 cell lines that express biologically active adenovirus type 5 precursor terminal protein (pTP) have been made. The amount of pTP synthesized in these cell lines ranges from barely detectable to greater than that observed in cells infected with the wild-type virus. The pTP-expressing cell lines permit the growth of a temperature-sensitive terminal protein mutant virus sub100r at the nonpermissive temperature. A higher percentage of the stably transfected 293 cell lines expressed terminal protein, and generally at considerably higher levels, than did the HeLa cell lines. While 293 cells appeared to tolerate pTP better than did HeLa cells, high-level pTP expression in 293 cells led to a significantly reduced growth rate. The 293-pTP cell lines produce infectious virus after transfection with purified viral DNA and form plaques when overlaid with Noble agar after infection at low multiplicity. These cell lines offer promise for the production of adenoviruses lacking pTP expression and therefore completely defective for replication.