Candidate partitioning genes (parA and parB) for the linear chromosome of Streptomyces coelicolor were identified by DNA sequencing in a series of seven genes located between rnpA and trxA near the chromosomal replication origin. The most likely translation start point of parB overlapped the parA stop codon, suggestive of coregulation, and transcription analysis suggested that the two genes formed an operon. Deletion of part of parB had no effect on the growth or appearance of colonies but caused a deficiency in DNA partitioning during the multiple septation events involved in converting aerial hyphae into long chains of spores. At least 13% of spore compartments failed to inherit the normal DNA allocation. The same phenotype was obtained with a deletion removing a segment of DNA from both parA and parB. Reinforcing the idea of a special role for the par locus during sporulation, the stronger of two parAB promoters was greatly upregulated at about the time when sporulation septation was maximal in colonies. Three copies of a 14-bp inverted repeat (GTTTCACGT GAAAC) were found in or near the parAB genes, and at least 12 more identical copies were identified within 100 kb of oriC from the growing genome sequence database. Only one perfect copy of the 14-bp sequence was present in approximately 5 Mb of sequence available from the rest of the genome. The 14-bp sequence was similar to sequences identified as binding sites for Spo0J, a ParB homologue from Bacillus subtilis believed to be important for DNA partitioning

The origins of life likely required the cooperation among a set of molecular species interacting in a network. If so, then the earliest modes of evolutionary change would have been governed by the manners and mechanisms by which networks change their compositions over time. For molecular events, especially those in a pre-biological setting, these mechanisms have rarely been considered. We are only recently learning to apply the results of mathematical analyses of network dynamics to prebiotic events. Here, we attempt to forge connections between such analyses and the current state of knowledge in prebiotic chemistry. Of the many possible influences that could direct primordial network, six parameters emerge as the most influential when one considers the molecular characteristics of the best candidates for the emergence of biological information: polypeptides, RNA-like polymers, and lipids. These parameters are viable cores, connectivity kinetics, information control, scalability, resource availability, and compartmentalization. These parameters, both individually and jointly, guide the aggregate evolution of collectively autocatalytic sets. We are now in a position to translate these conclusions into a laboratory setting and test empirically the dynamics of prebiotic network evolution.

ABSTRACT:The purposes of this study were to (a) gain an understanding of the views of inquiry held by faculty members involved in undergraduate science teaching and (b) describe the challenges, constraints, and opportunities that they perceived in designing and teaching inquiry-based laboratories. Participants included 19 college professors, representing both life and physical science disciplines, from (a) 2-year community college, (b) small, private nonprofit liberal arts college, (c) public master's granting university, and (d) public doctoral/research extensive university. We collected data through semistructured interviews and applied an iterative data analysis process. College science faculty members held a "full and open inquiry" view, seeing classroom inquiry as time consuming, unstructured, and student directed. They believed that inquiry was more appropriate for upper level science majors than for introductory or nonscience majors. Although faculty members valued inquiry, they perceived limitations of time, class size, student motivation, and student ability. These limitations, coupled with their view of inquiry, constrained them from implementing inquiry-based laboratories. Our proposed inquiry continuum represents a broader view of inquiry that recognizes the interaction between two dimensions of inquiry: (a) the degree of inquiry and (b) the level of student directedness, and provides for a range of inquiry-based classroom activities.

Phylogenetic-comparative and mutational analyses were used to elucidate the structure of the catalytically active RNA component of eubacterial ribonuclease P (RNase P). In addition to the refinement and extension of known structural elements, the analyses revealed a long-range interaction that results in a second pseudoknot in the RNA. This feature strongly constrains the three-dimensional structure of RNase P RNA near the active site. Some RNase P RNAs lack this structure but contain a unique, possibly compensating, structural domain. This suggests that different RNA structures located at different positions in the sequence may have equivalent architectural functions in RNase P RNA.

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Galactose utilization in Streptomyces lividans was shown to be controlled by an operon that is induced in the presence of galactose and repressed by glucose. Two promoters, gaIPi and galP2, which direct transcription of two distinct polycistronic transcripts, have been identified. galPI is located immediately upstream of the operon and is induced in the presence of galactose. This promoter directs transcription of the gaiT, gaIE, and galK genes. The second promoter, gaLP2, is located within the operon just upstream of the gaIE gene. This promoter is responsible for constitutive transcription of the galE and galK genes. Comparison of the S. lividans gal operon to the Escherichia coli gal operon indicates the presence of a constitutive promoter positioned upstream of galE in both operons. We suggest that coupling the operon's constitutive promoter to the galE gene fulfills a physiological requirement for constitutive UDPgalactose 4-epimerase expression in Streptomyces.The coordinate activation of sets of genes often involves complex combinations of regulatory signals. Many basic concepts about the mechanisms of gene expression in bacteria were formulated from work with the carbon catabolic and amino acid biosynthetic operons of Escherichia coli (1, 2). There are, however, many organisms that utilize different regulatory mechanisms to accomplish the same metabolic events. For example, the genes responsible for galactose utilization in E. coli are organized within a polycistronic operon. The operon is transcribed from two overlapping promoters, PI and P2. The PI promoter is positively activated by a cAMP-receptor protein, whereas P2 is repressed upon PI activation. Both promoters are negatively regulated by the gal repressor (3). The galactose utilization genes of Saccharomyces cerevisiae are also clustered and coordinately expressed. However, each gene is transcribed from its own promoter. The promoters are negatively controlled by the gal80 gene product and positively activated by the gal4 gene product (4). As in E. coli, galactose utilization in Sa. cerevisiae is subject to catabolite repression. Catabolite repression in Sa. cerevisiae, however, is not mediated by cAMP but by the hexokinase isoenzyme PII (5, 6). Thus, the processes that control gene activation reflect both the unique biology of individual organisms and universal principles of gene expression.We are interested in gene regulation in the Gram positive, differentiating bacterium Streptomyces. Members of this genus express a variety of interesting gene sets including those responsible for its morphological differentiation (7) and the biosynthesis of antibiotics (8, 9). We have chosen to study the galactose utilization operon as an example of a regulated set of genes in Streptomyces lividans because it is subject to glucose repression (10) and galactose induction (11). This paper describes the isolation and characterization of the promoters that direct transcription of the St. lividans gal operon. We demonstrate that the St. lividans galactose ...

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A 23-kb fragment of the Streptomyces coelicolor chromosome spanning the dnaA region has been isolated as a cosmid clone. Nucleotide sequence analysis of a 5-kb portion shows that the genes for the RNase P protein (rnpA), ribosomal protein L34 (rpmH), the replication initiator protein (dnaA4), and the beta subunit of DNA polymerase III (dnaN) are present in the highly conserved gene arrangement found in all eubacterial genomes studied so far. The dnaA-dnaN intergenic region is approximately 1 kb and contains a cluster of at least 12 DnaA boxes with a consensus sequence of TTGTCCACA matching the consensus DnaA box in the phylogenetically related Micrococcus luteus. Two DnaA boxes precede the dnaA sequence. We propose that the chromosomal origin (oriC) of S. coelicolor lies between dnaA and dnaN. In related work, J. ZakrzewskaCzerwinska and H. Schrempf (J. Bacteriol. 174:2688Bacteriol. 174: -2693Bacteriol. 174: , 1992 have identified the homologous sequence from the closely-related Streptomyces lividans as capable of self-replication.The genus Streptomyces represents a group of grampositive soil bacteria containing DNA with a high (approximately 73%) G+C content (1). The remarkable features of this group include the ability to elaborate a vast array of structurally diverse secondary metabolites and to undergo a complex cycle of mycelial growth and sporulation (for reviews, see references 4, 5, and 15). In recent years, considerable progress has been made in identifying genes involved in primary metabolism, antibiotic production, morphological differentiation, and the regulation of these processes. In contrast, some of the fundamental aspects of the flow of genetic information have not been addressed at a molecular level. This is especially true of DNA replication in these organisms. Little is known about the cellular machinery and regulatory events that are required to initiate and complete the synthesis of plasmid molecules, bacteriophage genomes, or the chromosome.It is known that in Streptomyces coelicolor, the most genetically characterized member of the genus, all of the essential genetic information is present on a single circular chromosome of approximately 8 Mb (13). Since, in other bacteria, chromosome replication begins at a unique sequence (the origin, oriC; see reference 28 for a review) and it is this site that may be a target for regulatory proteins (12), we decided to clone the oriC homolog from S. coelicolor. To do this, we took advantage of the striking conservation of gene arrangement that was apparent when the oriC regions of Pseudomonas putida, Bacillus subtilis, and Micrococcus luteus were compared (7,21). Thus, the genes for ribosomal protein L34 (rpmH), the protein subunit of RNase P (mpA), the replication initiator protein (dnaA), and the beta subunit of DNA polymerase III (dnaN) were present in the same order and relative orientation in each genome, and oriC was adjacent to dnaA. Since, in a previous study, we had isolated the rpmH-rnpA gene cluster from a streptomycete (19), this was used ...