Drosophila ARSs contain the yeast ARS consensus sequence and a replication enhancer.

A number of restriction fragments that function as autonomously replicating sequences (ARSs) in yeast have been isolated from Drosophila melanogaster DNA. The behaviour in yeast of plasmids containing Drosophila ARS elements was studied and compared to that exhibited by the archetypal yeast ARS-1 plasmid. ARS functions were localised by subcloning and BAL-31 deletion analysis. These studies demonstrated the structural and functional complexity of Drosophila ARSs. Each Drosophila ARS element has at least two domains, one essential for replication (the replication sequence, RS) and a second (the replication enhancer, RE) which is essential for maximum function of the RS. The RS of three Drosophila ARSs was shown to contain a sequence identical to an 11 bp yeast ARS consensus sequence (5' A/T TTTATPuTTT A/T 3'). These observations lend support to the hypothesis that heterologous ARS elements may be of biological significance.     

Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus

Eukaryotic chromosomes possess multiple origins of replication, whereas bacterial chromosomes are replicated from a single origin. The archaeon Pyrococcus abyssi also appears to have a single origin, suggesting a common rule for prokaryotes. However, in the current work, we describe the identification of two active origins of replication in the single chromosome of the hyperthermophilic archaeon Sulfolobus solfataricus. Further, we identify conserved sequence motifs within the origins that are recognized by a family of three Sulfolobus proteins that are homologous to the eukaryotic initiator proteins Orc1 and Cdc6. We demonstrate that the two origins are recognized by distinct subsets of these Orc1/Cdc6 homologs. These data, in conjunction with an analysis of the levels of the three Orc1/Cdc6 proteins in different growth phases and cell cycle stages, lead us to propose a model for the roles for these proteins in modulating origin activity.     

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.     

Identification and sequence of the basic replication region of a broad-host-range plasmid isolated from Thiobacillus ferrooxidans.

The minimum region required for replication of the broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2 in Escherichia coli was shown to be contained on a 2.05-kilobase fragment of DNA. A 184-base-pair fragment that was required in cis for plasmid replication was identified. This region was also involved in plasmid incompatibility. Nucleotide sequencing of this region revealed three perfectly conserved 22-base-pair tandemly repeated sequences. A comparison of this region with the equivalent region of the broad-host-range plasmid R1162 showed that the repeated sequences had 60% nucleotide homology. The 106-base-pair region immediately adjacent to the repeated sequences was 75% homologous. These plasmids were compatible.     

Conservation value of secondary forest habitats for endemic birds,a perspective from two widely separated tropical ecosystems

Tropical secondary forests are increasingly widespread, but their potential for conserving endemic birds remains unclear. Previous studies report different results; however all have been restricted to geographically discreet locations. This is important as different ecosystems are influenced by different external factors, possibly influencing conservation potential. Here we use consistent survey methods to examine how endemic bird richness varies between primary and secondary forest habitats in two widely separated tropical ecosystems, providing a more global context for evaluating the conservation value of secondary forests. Research was completed in Lambusango Forest Reserve (LFR) on Buton Island, Sulawesi, and Cusuco National Park (CNP), a Honduran cloud forest reserve. Bird communities in both forests were surveyed using 50 m radius point counts. Vulnerability assessments based on ecological theory on avifaunal assemblages were then conducted, which suggested endemics in LFR to be more susceptible to disturbance than those in CNP. Contrary to the results from our vulnerability assessments, endemics in CNP were less tolerant of moderate habitat modification than those in LFR. Richness of Mesoamerican endemics per study site declined significantly between core zone forest (6.34 ± 0.81) and more degraded forest in the boundary zone (3.86 ± 0.69). Richness of Wallacean endemics was similar in primary (4.89 ± 1.68) and disturbed secondary forest (4.52 ± 1.62). We recommend considering local and regional biogeographical and ecological factors when determining the conservation value of secondary forests, and suggest examples of potential importance, including differential community richness, influence of figs and human settlement patterns.     

The majority of minicircle DNA in Crithidia fasciculata strain CF-C1 is of a single class with nearly homogeneous DNA sequence

DNA minicircles found within the kinetoplast of the trypanosomatid Crithidia fasciculata, like those of most other kinetoplastid species, are heterogeneous in sequence. The pattern of minicircle DNA fragments generated by cleavage of kinetoplast DNA with various restriction enzymes has been used to demonstrate this heterogeneity. Here we describe a strain of Crithidia fasciculata in which more than 90% of the DNA minicircles exhibit a common pattern of restriction enzyme cleavage sites. A map of cleavage sites within this major minicircle DNA class is presented for seven restriction enzymes with hexanucleotide recognition sequences. Sequence homogeneity at an even finer level is reflected in minicircle DNA digestion patterns generated by restriction enzymes with tetranucleotide recognition sites. Partial DNA sequence analysis of multiple clones from the major minicircle class shows nearly complete homogeneity at the nucleotide level. The existence of a near homogeneous complement of DNA minicircles in Crithidia should facilitate the study of their replication in this organism.     

A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.

Replication of the kinetoplast DNA minicircle light strand initiates at a highly conserved 12-nucleotide sequence, termed the universal minicircle sequence. A Crithidia fasciculata single-stranded DNA-binding protein interacts specifically with the guanine-rich heavy strand of this origin-associated sequence (Y. Tzfati, H. Abeliovich, I. Kapeller, and J. Shlomai, Proc. Natl. Acad. Sci. USA 89:6891-6895, 1992). Using the universal minicircle sequence heavy-strand probe to screen a C. fasciculata cDNA expression library, we have isolated two overlapping cDNA clones encoding the trypanosomatid universal minicircle sequence-binding protein. The complete cDNA sequence defines an open reading frame encoding a 116-amino-acid polypeptide chain consisting of five repetitions of a CCHC zinc finger motif. A significant similarity is found between this universal minicircle sequence-binding protein and two other single-stranded DNA-binding proteins identified in humans and in Leishmania major. All three proteins bind specifically to single-stranded guanine-rich DNA ligands. Partial amino acid sequence of the endogenous protein, purified to homogeneity from C. fasciculata, was identical to that deduced from the cDNA nucleotide sequence. DNA-binding characteristics of the cDNA-encoded fusion protein expressed in bacteria were identical to those of the endogenous C. fasciculata protein. Hybridization analyses reveal that the gene encoding the minicircle origin-binding protein is nuclear and may occur in the C. fasciculata chromosome as a cluster of several structural genes.     

A structural analysis of the bent kinetoplast DNA from Crithidia fasciculata by high resolution chemical probing.

The chemical probes potassium permanganate (KMnO4) and diethylpyrocarbonate (DEPC) have been used to study the conformation of bent kinetoplast DNA from Crithidia fasciculata at different temperatures. Chemical reactivity data shows that the numerous short A-tracts of this bent DNA adopt a similar structure at 43 degrees C. This conformation appears to be very similar to the conformation of A-tracts in DNA exhibiting normal gel mobility. The A-tract structure detected by chemical probing is characterized by a high degree of base stacking on the thymine strand, and by an abrupt conformational change at the 3' end of the adenine strand. In general, no major alteration of this A-tract specific structure was detected between 4-53 degrees C. However, probing with KMnO4 revealed two unusual features of the C. fasciculata sequence that may contribute to the highly aberrant gel mobility of this DNA: 1) the B DNA/A-tract junction 5' dC/A3-6 3'. 5' dT3-6/G 3' is disproportionately represented and is conformationally distinct from other 5' end junctions, and 2) low temperature favors a novel strand-specific conformational distortion over a 20 base pair region of the bent kinetoplast DNA. Presence of the minor groove binding drug distamycin had little detectable effect on the A-tract conformation. However, distamycin did inhibit formation of the novel KMnO4 sensitive low temperature structure and partially eliminated the anomalous gel mobility of the kinetoplast DNA. Finally, we describe a simple and reproducible procedure for the production of an adenine-specific chemical DNA sequence ladder.     

Identification of the DNA sequence from the E. coli terminus region that halts replication forks

The terminus region of the E. coli chromosome contains two loci, T1 and T2, that inhibit the progress of replication forks and require the trans-acting factor tus. We have identified a 23 bp terminator signal at T1 and T2 that is within 100 bp of the sites of replication arrest. When an oligodeoxyribonucleotide containing the terminator signal was inserted into a plasmid, replication was halted only in a tus+ strain and when the terminator signal was oriented properly. We also found this terminator sequence in the terminus region of the plasmid R6K and in the origin region of RepFIIA class plasmids. In addition, we found striking similarities between the E. coli terminator signal and the terminator sequence of B. subtilis.     

A unique endonuclease from Crithidia fasciculata which recognizes a bend in the DNA helix. Specificity of the cleavage reaction

The introduction of a single nick in DNA circles by Crithidia fasciculata nicking enzyme (Shlomai, J., and Linial, M. (1986) J. Biol. Chem. 261, 16219-16225) requires the presence of a bent structure in the DNA helix. However, the sequence directing the local bending of the DNA helix is not per se a preferred site for nicking by the enzyme. No extensive sequence specificity is involved in defining the cleavage site for C. fasciculata nicking enzyme in the duplex circular DNA substrate. However, the abundance of A and T residues is significantly high at both the 3' and the 5' termini generated at the nicked site. Nicking of the sequence-directed bent fragment from C. fasciculata kinetoplast DNA minicircles correlates with the periodicity determined by the unique nucleotide distribution in the bent sequence, reflected in its thermodynamic parameters. Occurrence of nicking is best correlated with the predicted minima of the melting temperature and delta G profiles, as well as with A and T dinucleotide sequences at the nicked site, in both the supercoiled and the relaxed sequence-directed bent DNA substrates. The potential role of the bend-dependent nicking reaction in the replication of kinetoplast DNA minicircles is discussed.     

Characterization of a phage-plasmid hybrid (phasyl) with two independent origins of replication isolated from Escherichia coli.

The phage-plasmid hybrid phasyl can replicate as a phage in the presence of a filamentous phage of Escherichia coli (M13, fl, fd). The extragenic region of phasyl shows homology with the plus and the minus origins of filamentous phages. Insertion of a Cmr fragment into the plus origin or of a Kmr fragment into the minus origin resulted in a reduced transduction frequency, while insertion into other parts of the extragenic region did not. This suggests that phagelike replication of phasyl is mediated by an origin that coincides with the two homologous elements in the extragenic region. Autonomous replication of phasyl occurs from a second origin (oriA) that is located between positions 297 and 636. This fragment mediates replication if the Arp protein is supplied in trans. Arp is the only phage-encoded protein and is essential for plasmidlike replication. No sequence homology to other known origins was found. Phasyl derivatives with either one of the two origins inactivated can be rescued via the alternative replication mode, suggesting that the two replication pathways are independent.     

Construction of a viable SV40 variant containing two functional origins of DNA replication.

Viable variants of simian virus 40 (SV40) have been constructed which contain two functional origins of DNA replication (Or). The variants were made by introducing, at 0.175 on the SV40 map, a segment of DNA containing the viral Or. Two types of experiments demonstrate that the second Or is functional. First, the distribution of radioactivity in pulse-labeled SV40 (I) DNA is dramatically altered in the variants when compared with the parental virus. Second, electron microscopic examination of viral replicative intermediates indicates that while there is one initiation site for DNA synthesis in the parental genome, there are two sites in the variant. It was possible to introduce a deletion which inactivated the original Or at 0.67 map units in this variant. The resulting mutant could be propagated, and its DNA replication originated at the site of the newly inserted Or.