Comparison of the two major ARS elements of the ura4 replication origin region with other ARS elements in the fission yeast,Schizosaccharomyces pombe

We have previously peported that the replication orgin region located near the ura4 gene on chromosome III of the fission yeast, Schizosaccharomyces pombe, contains three closely spaced origins, each associated with an autonomously replicating sequence (ARS) element. Here we report the nucleotide sequences of two of these ARS elements, ars3002 and ars3003. The two ARS elements are located on either side of a transcribed 1.5 kb open reading frame. Like 11 other S. pombe ARS elements whose sequences have previously been determined in other laboratories, the 2 new ARS elements are unusually A+T-rich. All 13 ARS elements contain easily unwound stretches of DNA. Each of the ARS elements contains numerous copies, at a higher than expected frequency, of short stretches of A+T-rich DNA in which most of the Ts are on one strand and most of the As are on the complementary strand. We discuss the potential significance for ARS function of these multiple asymmetric A+T-rich sequences.     

Bending of DNA segments with Saccharomyces cerevisiae autonomously replicating sequence activity, isolated from basidiomycete mitochondrial linear plasmids

Summary Previous studies have indicated that DNA bending is a general structural feature of sequences (ARSs) from cellular DNAs of yeasts and nuclear and mitochondrial genomic DNAs of other eukaryotes that are capable of autonomous replication in Saccharomyces cerevisiae. Here we showed that bending activity is also tightly associated with S. cerevisiae ARS function of segments cloned from mitochondrial linear DNA plasmids of the basidiomycetes Pleurotus ostreatus and Lentinus edodes. Two plasmids, designated pLPO2-like (9.4 kb), and pLPO3 (6.6 kb) were isolated from a strain of P. ostreatus. A 1029 by fragment with high-level ARS activity was cloned from pLPO3 and it contained one ARS consensus sequence (A/T)TTTAT(A/G)TTT(A/T) indispensable for activity and seven dispersed ARS consensus-like (10/11 match) sequences. A discrete bent DNA region was found to lie around 500 by upstream from the ARS consensus sequence (T-rich strand). Removal of the bent DNA region impaired ARS function. DNA bending was also implicated in the ARS function associated with a 1430 by fragment containing three consecutive ARS consensus sequences which had been cloned from the L. edodes plasmid pLLE1 (11.0 kb): the three consecutive ARSs responsible for high-level ARS function occurred in, and immediately adjacent to, a bent DNA region. A clear difference exists between the two plasmid-derived ARS fragments with respect to the distance between the bent DNA region and the ARS consensus sequence(s).     

Single-strand-binding factor(s) which interact with ARS1 of Saccharomyces cerevisiae

Since plasmids containing autonomously replicating sequence(s) (ARS) can transform Saccharomyces cerevisiae cells at high frequency, ARS are considered to be the replication origins of chromosomes. To study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors which interact with the ARS1 region located near the centromere of chromosome IV in S. cerevisiae. Using the gel-shift assay, we found protein factors which bound to a single-stranded, 97-bp fragment of the ARS1 region containing the core consensus. Competition experiments with various oligodeoxyribonucleotides (oligos) suggest that a site recognized by the factor(s) was within the element containing the core consensus and adjacent close matches to the core consensus of the minus strand. Indeed, when the oligo containing the minus strand of this element was used as a probe, two oligo-protein complexes were detected. Mutations in the core consensus reduced these binding activities. When the plus-strand oligo of the same region was used as a probe, a retarded band was also detected, but with less specific binding. Considering the fact that the core consensus and close matches to the core consensus are important for ARS function, these results imply that the protein factors detected in this experiment may participate in DNA replication.     

The yeast protein encoded by PUB1 binds T-rich single stranded DNA.

We have characterized binding activities in yeast which recognise the T-rich strand of the yeast ARS consensus element and have purified two of these to homogeneity. One (ACBP-60) is detectable in both nuclear and whole cell extracts, while the other (ACBP-67) is apparent only after fractionation of extracts by heparin-sepharose chromatography. The major binding activity detected in nuclear extracts was purified on a sequence-specific DNA affinity column as a single polypeptide with apparent mobility of 60kDa (ACBP-60). This protein co-fractionates with nuclei, is present at several thousand copies per cell and has a Kd for the T-rich single strand of the ARS consensus between 10(-9) and 10(-10) M. Competition studies with simple nucleic acid polymers show that ACBP-60 has marginally higher affinity for poly dT30 than for a 30 nt oligomer containing the T-rich strand of ARS 307, and approximately 10 fold higher affinity for poly rU. Internal sequence information of purified p60 reveals identity with the open reading frames of genes PUB1 and RNP1 which encode polyuridylate binding protein(s). The second binding activity, ACBP-67, also binds specifically to the T-rich single strand of the ARS consensus, but with considerably lower affinity than ACBP-60. Peptide sequence reveals that the 67kDa protein is identical to the major polyA binding protein in yeast, PAB1.     

Identification and purification of a protein that binds the yeast ARS consensus sequence

We have identified a yeast protein that binds specifically to the ARS consensus sequence. By two-step chromatography we have purified the factor to apparent homogeneity as a single polypeptide of 67 kd. The purified ARS consensus-binding protein (ACBP) recognizes the ARS consensus of the four genomic ARS elements tested, binding preferentially to the T-rich single strand. Point mutations in the consensus significantly reduce the affinity of the single-strand binding. At the histone H4 ARS, ACBP recognizes both the perfect ARS consensus and a 9/11 match 3' of it. These two binding sites correlate with the boundaries of the minimal functional H4 ARS element. A similar configuration of binding sites is found at ARS1. We propose a model implicating this factor in an early step of the initiation of DNA replication.     

A single-stranded DNA binding protein from S. cerevisiae specifically recognizes the T-rich strand of the core sequence of ARS elements and discriminates against mutant sequences.

A protein named ssARS-T binding protein has been purified from yeast that specifically binds to the T-rich strand of the consensus core sequence of yeast autonomously replicating sequence (ARS) elements. As assayed from gel mobility shift experiments the ssARS-T protein shows characteristics of a sequence specific single-stranded DNA binding protein. The complementary A-rich strand of the ARS core sequence is bound much more weakly and no binding can be detected for the double-stranded form of the core sequence. Three single base substitutions in the core sequence that are known to abolish ARS function in vivo also lead to weaker binding of the core sequence to the ssARS-T protein in vitro. The strong correlation between the binding of mutated sequences in vitro and the ARS properties of these sequences in vivo points to an essential function of the ssARS-T protein during replication initiation in yeast ARS elements.     

The complete mitochondrial genome of the leafminer <Emphasis Type="Italic">Liriomyza trifolii</Emphasis> (Diptera: Agromyzidae)

Liriomyza trifolii (Diptera: Agromyzidae) is one of the most economically significant pests in the world. In this paper we present sequence data for the complete mitochondrial genome of L. trifolii. The circular genome is 16,141 bp long and contains one encoding region including 37 genes and one non-coding A+T-rich region. Gene numbers and organization is similar to that of the typical insect mitochondrial genomes except that two additional tRNA genes are found in the A+T-rich region (tRNA^Thr and tRNA^Leu(UUR)). All of the protein initiation codons are ATN, except ND1 which begins with GTG and COI which is initiated by the quadruplet ATCA. The 22 tRNA anticodons of L. trifolii match those observed in Drosophila yakuba, and all of tRNAs form the typical cloverleaf structure except for tRNA^Ser(AGN), which has lost the DHU-arm. The A+T-rich region of L. trifolii also contains two previously noted Diperan features—a highly conserved polyT stretch and a (TA)_n stretch.     

A yeast replication origin consists of multiple copies of a small conserved sequence

Autonomously replicating sequences (ARSs) of the yeast S. cerevisiae function as replication origins on plasmids and probably also on chromosomes. ARS function requires a copy of the ARS core consensus (5'-[A/T]TTTAT[A/G]TTT[A/T]-3') and additional sequences 3' to the T-rich strand of the consensus. Our analysis of an ARS from chromosome III, the C2G1 ARS, suggests that ARS function depends on the presence of an exact match to the core consensus and the presence of additional near matches in the 3' flanking region. We have demonstrated that ARS function can be mediated by multiple matches to the core consensus by constructing synthetic ARS elements from oligonucleotides containing copies of the consensus sequence. We find that two copies of the core consensus are sufficient for ARS activity and that an artificial ARS as efficient as a natural chromosomal ARS can be constructed from multiple core consensus elements in a specific orientation.     

HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter

Gel retardation assays using pea nuclear extracts have detected specific binding to regions of the promoter of the pea plastocyanin gene (petE). Several complexes which differ in sensitivity to competition with unlabelled promoter fragments and various DNA alternating copolymers, to heat treatment and to digestion with proteinase K have been detected. A protein factor, PCF1, forming one of these complexes was heat-stable and most sensitive to competition with poly(dAdT).poly(dAdT) compared to other alternating copolymers. DNase I footprinting assays showed that tracts of A/T-rich sequence within the -444 to -177 positive regulatory region of the petE promoter were protected in the presence of the pea nuclear extract. The factor PCF1 copurified with a high-mobility-group (HMG) protein preparation from pea chromatin. DNase I footprinting with the HMG protein preparation demonstrated that similar tracts of A/T-rich sequences within the promoter were protected. Southwestern-blot analysis of pea HMG proteins purified by gel filtration through Superose 12 detected a single DNA-binding species of 21 kDa. The properties of the factor PCF1 suggest that it is likely to be an HMG I protein.     

Mutational analysis of the consensus sequence of a replication origin from yeast chromosome III.

Yeast autonomously replicating sequence (ARS) elements contain an 11-base-pair core consensus sequence (5'-[A/T]TTTAT[A/G]TTT[A/T]-3') that is required for function. The contribution of each position within this sequence to ARS activity was tested by creating all possible single-base mutations within the core consensus sequence of ARS307 (formerly called the C2G1 ARS) and testing their effects on high-frequency transformation and on plasmid stability. Of the 33 mutations, 22 abolished ARS function as measured by high-frequency transformation, 7 caused more than twofold reductions in plasmid stability, and 4 had no effect on plasmid stability. Mutations that reduced or abolished ARS activity occurred at each position in the consensus sequence, demonstrating that each position of this sequence contributes to ARS function. Of the four mutations that had no effect on ARS activity, three created alternative perfect matches to the core consensus sequence, demonstrating that the alternate bases allowed by the consensus sequence are, indeed, interchangeable. In addition, a change from T to C at position 6 did not perturb wild-type efficiency. To test whether the essential region extends beyond the 11-base-pair consensus sequence, the effects on plasmid stability of point mutations one base 3' to the T-rich strand of the core consensus sequence (position 12) and deletion mutations that altered bases 5' to the T-rich strand of the core consensus sequence were examined. An A at position 12 or the removal of three T residues 5' to the core consensus sequence severely diminished ARS efficiency, showing that the region required for full ARS efficiency extends beyond the core consensus sequence in both directions.     

The c-terminal DNA-binding domain ofChironomus BR gene products shows preferential affinity for (dA,dT)-rich sequences

Balbiani ring genes (BRs), the most active loci in the polytene chromosomes of the salivary gland of the midgeChironomus (Diptera), code for secretory giant peptides (the sp-I family). Evidence previously reported indicated that the conserved C-terminal region of proteins of the sp-I family had DNA-binding properties (assayed with sp-Ia), and one such region, derived fromBR2.2, which codes for the product sp-Ib, might occur as a stable independent peptide, being transferred to the nucleus where it is detectable in the largeBRs (BR1 andBR2), among other structures, by immunostaining. Here, we show that the C-terminal portion of one of theBR gene products, expressed as a glutathione-S-transferase fusion protein shows preferential affinity for A.T-rich sequences and binds with varying affinity to restriction fragments of the A.T-rich BR1 promoter. The binding was inhibited by distamycin, suggesting that the interaction involves the minor groove of the DNA. Analysis of the promoter fragments by gel electrophoresis indicated that most appeared to present a conspicuous bend, as deduced from their anomalous electrophoretic mobilities. Furthermore, the affinity of the C-terminal domain for the different promoter fragments appeared to correlate with the degree of bending. Thus, the C-terminal domain might play a role in controlling gene expression by binding to A.T-rich sequences, including those of theBR genes.     

Use of a short A/T-rich cassette for enhanced expression of cloned genes inEscherichia coli

A short (43-bp) A/T-rich stretch of DNA located in The intergenic region between thebaiA2 andbaiF genes fromEubacterium sp. strain VPI 12708 was amplified by polymerase chain reaction (PCR) and inserted in front of the Shine-Dalgarno (SD) sequences of three inefficiently-expressedEubacterium sp. strain VPI 12708 genes cloned inEschcrichia coli plasmids. Insertion of this A/T-rich cassette increased gene expression in all cases tested. Deletion of part of the A/T-rich region from abaiF clone in pUC19 resulted in decreased gene expression. Synthesis of specific mRNA was increased with addition of the A/T-rich cassette to constructs containing thebaiC gene from Eubacterium sp. strain VPI 12708, but mRNA synthesis was not significantly changed in cells containing plasmid constructs with thebaiF andbaiG genes. Enhanced translation resulting from a decrease in mRNA secondary structure in the ribosome binding site region is discussed as a possible reason for increased gene expression with the A/T-rich cassette.     

The Mcm467 complex of Saccharomyces cerevisiae is preferentially activated by autonomously replicating DNA sequences

We have analyzed the role of single-stranded DNA (ssDNA) in the modulation of the ATPase activity of Mcm467 helicase of the yeast Saccharomyces cerevisiae. The ATPase activity of the Mcm467 complex is modulated in a sequence-specific manner and that the ssDNA sequences derived from the origin of DNA replication of S. cerevisiae autonomously replicating sequence 1 (ARS1) are the most effective stimulators. Synthetic oligonucleotides, such as oligo(dA) and oligo(dT), also stimulated the ATPase activity of the Mcm467 complex, where oligo(dT) was more effective than oligo(dA). However, the preference of a thymidine stretch appeared unimportant, because with yeast ARS1 derived sequences, the A-rich strand was as effective in stimulating the ATPase activity, as was the T-rich strand. Both of these strands were more effective stimulators than either oligo(dA)( )()or oligo(dT). The DNA helicase activity of Mcm467 complex is also significantly stimulated by the ARS1-derived sequences. These results indicate that the ssDNA sequences containing A and B1 motifs of ARS1, activate the Mcm467 complex and stimulate its ATPase and DNA helicase activities. Our results also indicate that the yeast replication protein A stimulated the ATPase activity of the Mcm467 complex.     

Narrow A/T-rich zones present at the distal 5′-flanking sequences of the zein genes Zc1 and Zc2 bind a unique 30 kDa HMG-like protein

Nuclear extracts from maize endosperm were used to investigate protein-DNA interactions in the 5-upstream region of the Zc1 and Zc2 genes. These genes encode for zeins of apparent molecular mass (MW_app) 16 and 28 kDa, respectively, which accumulate in the endosperm during seed maturation. Binding assays revealed specific binding of a nuclear protein to three A/T-rich elements, 0.9–1.0 kbp upstream from the initiation codon. One of these elements (41 bp, 88% A/T), present in Zc1, contained a 13 nucleotide duplication. The other two (28 bp, 86% A/T; 42 bp alternating A-T) are consecutive elements in Zc2. Competition experiments strongly suggest that the three elements bind to the same protein. Protein-DNA interaction was detected in endosperm nuclear extracts of 8 to 21 days after pollination (DAP), as well as in 25 DAP embryos and in different tissues from plantlets. The protein factor has an MW_app of ca. 30 kDa. This factor has properties suggesting it is an HMG-like protein. These results are consistent with a growing accumulation of data for a number of genes indicating that A/T-rich elements, located at distal and proximal zones of the 5-flanking sequences, interact with HMG-like proteins.     

Isolation, Characterization, and Molecular Cloning of a Protein (Abp2) That Binds to a Schizosaccharomyces pombe Origin of Replication (ars3002)

The autonomously replicating sequence (ARS) element ars3002 is associated with the most active replication origin within a cluster of three closely spaced origins on chromosome III of Schizosaccharomyces pombe. A 361-bp portion of ars3002 containing detectable ARS activity includes multiple near matches to the S. pombe ARS consensus sequence previously reported by Maundrell et al. (K. Maundrell, A. Hutchison, and S. Shall, EMBO J. 7:2203–2209, 1988). Using a gel shift assay with a multimer of an oligonucleotide containing three overlapping matches to the Maundrell ARS consensus sequence, we have detected several proteins in S. pombe crude extracts that bind to the oligonucleotide and ars3002. One of these proteins, ARS binding protein 1, was previously described (Abp1 [Y. Murakami, J. A. Huberman, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 93:502–507, 1996]). In this report the isolation, characterization, and cloning of a second binding activity, designated ARS binding protein 2 (Abp2), are described. Purified Abp2 has an apparent molecular mass of 75 kDa. Footprinting analyses revealed that it binds preferentially to overlapping near matches to the Maundrell ARS consensus sequence. The gene abp2 was isolated, sequenced, and overexpressed in Escherichia coli. The DNA binding activity of overexpressed Abp2 was similar to that of native Abp2. The deduced amino acid sequence contains a region similar to a proline-rich motif (GRP) present in several proteins that bind A+T-rich DNA sequences. Replacement of amino acids within this motif with alanine either abolished or markedly reduced the DNA binding activity of the mutated Abp2 protein, indicating that this motif is essential for the DNA binding activity of Abp2. Disruption of the abp2 gene showed that the gene is not essential for cell viability. However, at elevated temperatures the null mutant was less viable than the wild type and exhibited changes in nuclear morphology. The null mutant entered mitosis with delayed kinetics when DNA replication was blocked with hydroxyurea, and advancement through mitosis led to the loss of cell viability and aberrant formation of septa. The null mutant was also sensitive to UV radiation, suggesting that Abp2 may play a role in regulating the cell cycle response to stress signals.     

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.     

A Short Region from the LEU2 Gene of Saccharomyces cerevisiae Functions as an ARS in the Yeast Saccharomyces exiguus Yp74L-3

We examined the autonomously replicating sequence (ARS) activity of some fragments derived from the LEU2 region of Saccharomyces cerevisiae onto Saccharomyces exiguus Yp74L-3. A DNA fragment functioning as an ARS in S. exiguus, but not in S. cerevisiae, was shown to exist. The ARS activity for S. exiguus was reduced by the 2-μm plasmid origin of S. cerevisiae when both elements coexisted on a single circular plasmid. Analysis of ARS activity with the PCR products from the fragment revealed that the ARS-acting sequence was located in the 3′-terminal area of the transcribed region of the LEU2 gene of S. cerevisiae. It is suggested that the ARS recognition system in S. exiguus is significantly different from that of S. cerevisiae. Received: 16 June 1998 / Accepted: 3 August 1998