Enforcement of late replication origin firing by clusters of short G-rich DNA sequences

Previous studies in budding yeast suggested that the default firing time of most DNA replication origins is early in S phase and that origins can be forced to fire later by proximity to certain cis-acting sequences. However, these cis-acting sequences were not well defined. We have attempted to characterize cis-acting sequences that affect replication timing in the fission yeast. We identified a stretch of 200 bp that was sufficient to compel nearby origins to fire late. The 200-bp stretch was able to force an origin to fire late whether adjacent to the origin or approximately 800 bp away in opposite orientation. The stretch contains a cluster of three close matches to a G-rich, 10-bp late consensus sequence (LCS). The three LCS elements cooperate with each other and with other sequences within the 200-bp stretch to enforce late replication. Although only a few origins that fire in very late S phase have been identified in fission yeast, all of them are located close to a cluster of LCS elements.     

Identification of the minimal essential region for the replication origin of miniF plasmid

Summary The minimal replication origin of miniF plasmid was found to lie within a region of 217 bp in length. This region contains an AT cluster and the four 19 bp direct repeats responsible for incompatibility, termed incB. Its location coincides with that of ori2 of plasmid F, previously inferred to be the replication starting point by electron microscopic analysis (Eichenlaub et al. 1981).Abbreviations kb kilobase(s) - bp base pairs - Ap ampicillin - Tc tetracycline     

Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2

Summary A DNA sequence cosisting of 617 base pairs (bp) from the region of the origin of replication of the broad-host range plasmid RK2 has been determined. Included within this sequence is a 393 bp HpaII restriction fragment that provides a functional origin or replication when other essential RK2 specified functions are provided in trans. Also contained in this sequence is a region, distinguished functionally from the replication origin, which is involved in the expression of inc ₂ incompatibility, i.e., the ability of derivatives of RK2 to eliminate a resident RK2 plasmid. The 617 bp sequence includes eight 17 base pair direct repeats with 5 located within the region required for a functional replication origin and 3 within the region involved in inc ₂ incompatibility. In addition, a 40 bp region rich in A-T followed by a 60 bp stretch having a high G+C content is present. Deletion evidence indicates that the A-T rich and possibly the G+C regions are required for a functional replication origin. Based on the evidence contained in this and the preceding paper (Thomas et al. 1980 b) a model will be presented for the involvement of these specific sequences in the initiation of RK2 DNA replication, plasmid maintenance and plasmid incompatibility.     

Centromeric DNA from chromosome VI in Saccharomyces cerevisiae strains

The functional sequence from the centromere in chromosome VI ( CEN6 ) of Saccharomyces cerevisiae was narrowed down to a stretch of 500 bp by a Bal31 deletion approach. The DNA sequence in this region shows three long stretches, 40 bp, 96 bp, and 63 bp of 85% and more AT pairs and a pyrimidine purine bias in the individual single strands. We assume that the CEN6 functional sequences encompass these AT-rich stretches because this part shows striking similarities to sequence elements common to CEN3 and CEN11 DNA. A strain comparison revealed that CEN6 DNA sequences are confined to the Saccharomyces genus and probably only to the S. cerevisiae species. CEN6 is not highly conserved within S. cerevisiae strains because EcoRI and HindIII restriction site variants are found with high frequency.     

Genome nucleotide composition shapes variation in simple sequence repeats

Simple sequence repeats (SSRs) or microsatellites are a common component of genomes but vary greatly across species in their abundance. We tested the hypothesis that this variation is due in part to AT/GC content of genomes, with genomes biased toward either high AT or high CG generating more short random repeats that are long enough to enhance expansion through slippage during replication. To test this hypothesis, we identified repeats with perfect tandem iterations of 1-6 bp from 25 protists with complete or near-complete genome sequences. As expected, the density and the frequency are highly related to genome AT content, with excellent fits to quadratic regressions with minima near a 50% AT content and rising toward both extremes. Within species, the same trends hold, except the limited variation in AT content within each species places each mainly on the descending (GC rich), middle, or ascending (AT rich) part of the curve. The base usages of repeat motifs are also significantly correlated with genome nucleotide compositions: Percentages of AT-rich motifs rise with the increase of genome AT content but vice versa for GC-rich subgroups. Amino acid homopolymer repeats also show the expected quadratic relationship, with higher abundance in species with AT content biased in either direction. Our results show that genome nucleotide composition explains up to half of the variance in the abundance and motif constitution of SSRs.     

The human minisatellite consensus at breakpoints of oncogene translocations.

A reexamination of human minisatellite (hypervariable) regions following the cloning and sequencing of the new minisatellite, VTR1.1, revealed that many of these structures possessed a strongly conserved copy of the chi-like octamer, GC[A/T]GG[A/T]GG. In oncogene translocations apparently created by aberrant VDJ recombinase activity, this VTR octamer was often found within a few bases of the breakpoint (p less than 10(-10)). Three bcl2 rearrangements which occurred within 2 bp of one another were located precisely adjacent to this consensus; it defined the 5' border of that oncogene's major breakpoint cluster. Several c-myc translocations also occurred within 2 bp of this sequence. While the appearance of a chi-like element in polymorphic minisatellite sequences is consistent with a role promoting either recombination or replication slippage, the existence of such elements at sites of somatic translocations suggests chi function in site-specific recombination, perhaps as a subsidiary recognition signal in immunoglobulin gene rearrangement. We discuss the implications of these observations for mechanisms by which oncogene translocations and minisatellite sequences are generated.     

Temperature can reversibly modify the structure and the functional efficiency of ori sequences of the yeast mitochondrial genome

We have compared the suppressibility of three isonuclear spontaneous, cytoplasmic petite mutants of Saccharomyces cerevisiae, as measured at three temperatures, 23 degrees C, 28 degrees C and 33 degrees C. The three petites have mitochondrial genomes made up of repeat units which are about 400 bp in size, and carry an origin of replication, ori1. This ori sequence is intact in petite Z1, whereas it lacks GC cluster A in petite 26 and cluster A plus some contiguous nucleotides in petite 14. These deletions lead to the impossibility to form a stem-and-loop structure of the ori sequence, the 'A-B fold', which involves two GC clusters, A and B, and the nucleotides in between. Instead, a 'replacement fold', only involving AT base pairs, is feasible. In petites 14 and 26, suppressivity decreases when the temperature is raised from 28 degrees C to 33 degrees C, and increases when the temperature is lowered from 28 degrees C to 23 degrees C. In contrast, no changes are seen in petite Z1. These temperature effects correlate with the stability of the 'A-B fold' and the instability of the 'replacement folds'. Since suppressibility measures the replicative competitiveness of the petite genome relative to the wild-type genome, these results indicate that an environmental parameter, temperature, can reversibly affect the structure and the functional efficiency of ori sequences in vivo. The evolutionary implications of these findings are discussed.     

Mosaic structure of the small cryptic plasmid pKST23 from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Small plasmid pKST23 was isolated from sheep ruminal Escherichia coli population. Plasmid sequence was determined to be 2,779 bp in length and was found to have an overall 42 % of GC pairs. However, its sequence can be divided into two regions based on genetic composition and the GC content. It was found that the high GC region spanning approximately from nucleotide 1,300 to 2,750 was identical to a group of small Escherichia coli plasmids and encoded a putative replication protein identical to plasmid pKL1 Rep protein. The part with lower GC pairs seemed to be more specific as it showed no similarity to the GenBank database. Computational analysis revealed four open reading frames, two of which showed considerable homology to replication proteins. PCR primers targeting parts of the two different regions of plasmid pKST23 were used to assess the occurrence of related plasmids within ruminal E. coli population.     

Mapping 5'' termini of JC virus early RNAs.

Within its enhancer promoter region, the MAD-1 strain of JC virus (JCV) has two 98-base-pair tandem repeats, each containing a TATA box-like sequence. In the present study, polyadenylated early JCV mRNAs were isolated 5 or 29 days after infection of primary human fetal glial (PHFG) cells. By using S1 nuclease, the 5' termini of the early mRNAs were mapped to nucleotide position(s) (np) 122 through 125, which lies within an AT rich region (at np 113 through 127). In contrast, when JCV DNA was transcribed in vitro, we observed a single major cluster of 5' start sites at np 94 through 97, which is approximately 25 base pairs downstream from one of the TATA boxes. By day 5, the earliest time at which JCV RNA was detected, viral DNA replication had begun; it continued for at least an additional 20 days. Since more late than early RNA was present at 5 days postinfection, the early RNAs whose synthesis began at np 122 through 125 may be analogous to SV40 late early mRNA (Ghosh and Lebowitz, J. Virol. 40:224-240, 1981). However, we have not detected RNAs with 5' termini 25 to 30 bp downstream from the TATA box at earlier times. While JCV contains two identical TATA boxes, one in each of the 98-bp repeats, only the upstream TATA box functions as an early promoter element.     

The complete nucleotide sequence of a small cryptic plasmid from Lactobacillus plantarum

The complete nucleotide sequence of a cryptic plasmid isolated from a Lactobacillus plantarum strain has been determined. The plasmid, designated pC30i1, has a molecular size of 2140 bp and a GC content of 37%. The sequence contains one major open reading frame (ORF R) of 951 bp, encoding a basic polypeptide of 317 amino acids, and a molecular weight of 36,956. ORF R shows extensive sequence similarity with genes coding for replication-associated proteins in a group of gram-positive plasmids known to replicate via single-stranded intermediates (ssDNA plasmids), and a stretch of 9 amino acids in the translation of ORF R closely matches a conserved region in these proteins, as well as the active site of the phi X174 Rep protein. Sequences similar to the ssDNA plasmid origins of replication are also present in the pC30i1 sequence, strengthening the hypothesis that pC30i1 belongs to the ssDNA plasmid family. The other main feature of the pC30i1 sequence is a noncoding region consisting of 14 direct, imperfect repeats of a 17-bp sequence, which may have an incompatibility function.     

Nucleotide sequence involved in the replication of cloned yeast mitochondrial DNA

A fragment of yeast mitochondrial DNA, Alu B, has two subfragments, Alu B1 and Alu B2. They were each cloned and sequenced. The autonomously replicating function of the curtailed Alu B1 (342 bp) was defined within 186 bp. A GC-rich sequence identical to the oris sequence in the curtailed Alu B1 was unnecessary for its autonomously replicating function. The 186 bp sequence had an ATATAAAT sequence and the stem and loop structures. The base sequence of Alu B2 also contained the same octanucleotides, the stem and loop structures, one oris sequence and one unique GC cluster. Yeast transformants with cloned Alu B2 grew slowly. The cloned Alu B2 was enlarged in the yeast host concomitantly with compensation of the slow growth of the transformants.     

Site and sequence specificity of the daunomycin-DNA interaction

The site and sequence specificity of the daunomycin-DNA interaction was examined by equilibrium binding methods, by deoxyribonuclease I footprinting studies, and by examination of the effect of the antibiotic on the cleavage of linearized pBR322 DNA by restriction endonucleases PvuI and EcoRI. These three experimental approaches provide mutually consistent results showing that daunomycin indeed recognizes specific sites along the DNA lattice. The affinity of daunomycin toward natural DNA increases with increasing GC content. The quantitative results are most readily explained by binding models in which daunomycin interacts with sites containing two adjacent GC base pairs, possibly occurring as part of a triplet recognition sequence. Deoxyribonuclease I footprinting studies utilizing the 160 base pair (bp) tyrT DNA fragment and 61 and 53 bp restriction fragments isolated from pBR322 DNA further define the sequence specificity of daunomycin binding. Specific, reproducible protection patterns were obtained for each DNA fragment at 4 degrees C. Seven protected sequences, ranging in size from 4 to 14 bp, were identified within the tyrT fragment. Relative to the overall tyrT sequence, these protected sequences were GC rich and contained a more limited and distinct distribution of di- and trinucleotides. Within all of the protected sequences, a triplet containing adjacent GC base pairs flanked by an AT base pair could be found in one or more copies. Nowhere in the tyrT fragment did that triplet occur outside a protected sequence. The same triplet occurred within seven out of nine protected sequences observed in the fragments isolated from pBR322 DNA. In the two remaining cases, three contiguous GC base pairs were found. We conclude that the preferred daunomycin triplet binding site contains adjacent GC base pairs, of variable sequence, flanked by an AT base pair. This conclusion is consistent with the results of a recent theoretical study of daunomycin sequence specificity [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466]. Adriamycin and the beta-anomer of adriamycin produce the same qualitative pattern of protection as daunomycin with the tyrT fragment. Daunomycin inhibits the rate of digestion of pBR322 DNA by PvuI (recognition sequence 5'-CGATCG-3') to a greater extent than it does EcoRI (recognition sequence 5'-GAATTC-3'), a finding consistent with the conclusions derived from our footprinting studies. Our results, as a whole, are the clearest indication to date that daunomycin recognizes a specific DNA sequence as a preferred binding site.     

An archaeal chromosomal autonomously replicating sequence element from an extreme halophile, Halobacterium sp. strain NRC-1

We report on the identification and first cloning of an autonomously replicating sequence element from the chromosome of an archaeon, the extreme halophile Halobacterium strain NRC-1. The putative replication origin was identified by association with the orc7 gene and replication ability in the host strain, demonstrated by cloning into a nonreplicating plasmid. Deletion analysis showed that sequences located up to 750 bp upstream of the orc7 gene translational start, plus the orc7 gene and 50 bp downstream, are sufficient to endow the plasmid with replication ability, as judged by expression of a plasmid-encoded mevinolin resistance selectable marker and plasmid recovery after transformation. Sequences located proximal to the two other chromosomally carried haloarchaeal orc genes (orc6 and orc8) are not able to promote efficient autonomous replication. Located within the 750-bp region upstream of orc7 is a nearly perfect inverted repeat of 31 bp, which flanks an extremely AT-rich (44%) stretch of 189 bp. The replication ability of the plasmid was lost when one copy of the inverted repeat was deleted. Additionally, the inverted repeat structure near orc7 homologs in the genomic sequences of two other halophiles, Haloarcula marismortui and Haloferax volcanii, is highly conserved. Our results indicate that, in halophilic archaea, a chromosomal origin of replication is physically linked to orc7 homologs and that this element is sufficient to promote autonomous replication. We discuss the finding of a functional haloarchaeal origin in relation to the large number of orc1-cdc6 homologs identified in the genomes of all haloarchaea to date.     

Multiple Divergent ITS1 Copies Were Identified in Single Tomato Genome Using DGGE Analysis

The intra-genomic variation in the internal transcribed spacer (ITS) region has led to misleading conclusions in the evolutionary analysis of plants; understanding this variation is critical for correct evolutionary analysis based on ITS sequences. To reveal the ITS variation in tomato, entire copies of ITS1 sequences within tomato species were separated using denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. ITS1 copies varied significantly in sequence composition, but not in sequence length within the same tomato cultivar. DNA sequence similarity of the ITS1 copies was 77–100 %. Moreover, AT and GC contents in ITS1 copies from each tomato cultivar were significantly different, ranging from 50.4 to 64.3 % for GC and from 35.7 to 49.6 % for AT. However, the length variation of ITS1 was insignificant, ranging from 279 to 282 bp. Multiple copies of divergent ITS1 present in the tomato genome indicate that some copies may be paralogues. In conclusion, DGGE technique is a reliable and novel approach to reveal the entire ITS copy variation and the possible evolutionary relationship of tomato.     

Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae.

Two dimensional gel electrophoretic techniques were used to locate all functional DNA replication origins in a 22.5 kb stretch of yeast chromosome III. Only one origin was detected, and that origin is located within several hundred bp of an ARS element.     

Chromosomal Localization and Sequence Variation of 5S rRNA Gene in Five Capsicum Species

Chromosomal localization and sequence analysis of the 5S rRNA gene were carried out in five Capsicum species. Fluorescence in situ hybridization revealed that chromosomal location of the 5S rRNA gene was conserved in a single locus at a chromosome which was assigned to chromosome 1 by the synteny relationship with tomato. In sequence analysis, the repeating units of the 5S rRNA genes in the Capsicum species were variable in size from 278 bp to 300 bp. In sequence comparison of our results to the results with other Solanaceae plants as published by others, the coding region was highly conserved, but the spacer regions varied in size and sequence. T stretch regions, just after the end of the coding sequences, were more prominant in the Capsicum species than in two other plants. High G-C rich regions, which might have similar functions as that of the GC islands in the genes transcribed by RNA PolII, were observed after the T stretch region. Although we could not observe the TATA like sequences, an AT rich segment at -27 to -18 was detected in the 5S rRNA genes of the Capsicum species. Species relationship among the Capsicum species was also studied by the sequence comparison of the 5S rRNA genes. While C. chinense, C. frutescens, and C. annuum formed one lineage, C. baccatum was revealed to be an intermediate species between the former three species and C. pubescens.     

The AT-rich tract of the SV40 ori core: negative synergism and specific recognition by single stranded and duplex DNA binding proteins.

The SV40 origin of replication comprises a run of thymine and adenine residues. Integrity of this AT-rich sequence is known to be essential for replication. We set out to study whether or not these elements can work synergistically to sustain replication. Quite surprisingly, additional copies of the AT stretch linked to a functional SV40 ori core dramatically reduce its replication in Cosl cells, probably by creating some physical block. Interestingly, the same inhibiting effect can be observed with the addition in cis of the yeast ARS consensus, which is homologous to the SV40 AT stretch. This modulation is possibly due to the action of cellular factors that recognize either of the two sequences. In fact, we demonstrate the existence of factor(s) in Cosl crude nuclear extracts that in vitro can specifically bind to either of them. Moreover, we show that these sequence-specific factor(s) (MW about 50 kDa), named SOAP, recognize both single (T-rich strand) and double stranded forms of the AT tracts. Binding to single stranded AT stretches can be specifically inhibited by the corresponding duplex form, but not vice versa.