Sialylation of N-Carbohydrate Chains of Glycoproteins with Trans-Sialidase from Trypanosoma cruzi

2,3-Sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosoma cruzi is reported. Trans-sialidase (160 kDa, pI 5.35–5.65) and its catalytic fragment (70 kDa, pI 6.0–6.3) were isolated from T. ruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained its activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarin-labeled oligosaccharides with various numbers of antennas and for 2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.     

Dissection of a pollen-specific promoter from maize by transient transformation assays

We have previously reported the isolation and characterization of a gene (Zm 13) from Zea mays which shows a pollen-specific pattern of expression. Stably transformed tobacco plants containing a reporter gene linked to portions of the Zm 13 5 flanking region show correct temporal and spatial expression of the gene. Here we present a more detailed analysis of the 5 regions responsible for expression in pollen by utilizing a transient expression system. Constructs containing the -glucuronidase (GUS) gene under the control of various sized fragments of the Zm13 5 flanking region were introduced into Tradescantia and Zea mays pollen via high-velocity microprojectile bombardment, and monitored both visually and with a fluorescence assay. The results suggest that sequences necessary for expression in pollen are present in a region from –100 to –54, while other sequences which amplify that expression reside between –260 and –100. The replacement of the normal terminator with a portion of the Zm13 3 region containing the putative polyadenylation signal and site also increased GUS expression. While the –260 to –100 region contains sequences similar to other protein-binding domains reported for plants, the –100 to –54 region appears to contain no significant homology to other known promoter fragments which direct pollen-specific expression. The microprojectile bombardment of Tradescantia pollen appears to be a good test system for assaying maize and possibly other monocot promoter constructs for pollen expression.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Association of polymorphic markers of the lipid metabolism genes with diabetic polyneuropathy in type 1 diabetes mellitus

A possible association of the polymorphic markers 2/3/4 of the apolipoprotein E gene (APOE) and I /D of the apolipoprotein B gene (APOB) with diabetic polyneuropathy (DPN) was analyzed in patients with type 1 diabetes mellitus (T1DM) with (N=86) or without (N=94) clinical signs of DPN. The two groups did not differ significantly in allele and genotype frequencies of the 2/3/4 polymorphic marker of the APOE gene. Analysis of the allele and genotype frequency distributions of the I/D polymorphic marker of the APOB gene showed that risk of DPN is higher in carriers of allele I or genotype I/I (OR=1.66 and 2.01, respectively) and lower in carriers of allele D (OR=0.60). The results implicate the APOB gene, which codes for one of the major components of the lipid metabolism system, in DPN development in patients with T1DM.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 230–234.Original Russian Text Copyright © 2005 by Voronko, Yakunina, Strokov, Lavrova, Nosikov.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

Variation in Ambrosia pollen concentration in Southern and Central Poland in 1982–1999

The aim of the study was to investigate theAmbrosia pollen concentrations inselected Polish cities and for Kraków torelate it to some meteorological factors.Sampling was carried out in Kraków in1982–1997 and in Rabka in 1992–1996 with theuse of the gravimetric method. In Zakopane,Kraków, Ostrowiec witokrzyski,Warszawa and Pozna in 1995–1996 both thegravimetric and volumetric methods (Burkardtrap) were employed. In Kraków themonitoring has been performed since 1994 usingthe volumetric method. The results show theragweed pollen presence in August and Septemberwith the tendency to appear more frequently inAugust in some years. In Kraków (1994–1999)Ambrosia pollen was found either in thelast two weeks of August or in the first twoweeks of September which seems to be a regularand repeatable pattern every two years.Seasonal fluctuations of Ambrosia pollenconcentration do not show a clear increasingtendency except at Warszawa and Ostrowiecwitokrzyski in 1996 and at Krakówin 1999. Percentage of Ambrosia pollen inannual sums of total pollen is very low anddoes not exceed 1% except at Ostrowiecwitokrzyski in 1996 (1.2%) and atKraków in 1999 (2%). For Kraków theanalysis of some meteorological factors (Tmax,Tmin, precipitation, wind direction) wasperformed. High temperature and lack of rain orlow precipitation correlate well with ragweedpollen concentrations. During the Ambrosia pollen seasons ESE, E, S, SE, WSW, SWwind directions prevailed which could suggest along-distance transport from Ukraine, the CzechRepublic, Slovakia and also from Hungary, one ofthe most ragweed-polluted countries.     

Increased erythritol production in Torula sp. by Mn2+and Cu2+

The production of erythritol and the erythritol yield from glucose by Torula sp. were improved, in increasing order, by supplementing with 10 mg MnSO₄4H₂O l^–1, 2 mg CuSO₄5H₂O l^–1, and both 10 mg MnSO₄4H₂O l^–1 and 2 mg CuSO₄5H₂O l^–1. Mn²⁺ decreased the intracellular concentration of erythritol, whereas Cu²⁺ increased the activity of erythrose reductase in cells. These results suggest that Mn²⁺ altered the permeability of cells, whereas Cu²⁺ increased the activity of erythrose reductase in cells.     

Inheritance and linkage of isozyme loci in almond

The segregation of seven isozyme marker genes was investigated using eight controlled crosses in almond. The cultivar Nonpareil was the maternal parent in all crosses. Pollination was achieved using eight different cultivars, and a total of 3200 individual kernels were assessed. For each isozyme the goodness-of-fit test was used to test for departure from the expected frequencies assuming Mendelian inheritance. Given a higher than expected number of significant results for individual isozymes, independent segregation between pairs of isozymes was tested using the chi-square statistic on the resulting two-way contingency tables. In all crosses a highly significant association (P value< 0.001) was observed between (1) the AAT- 1 and IDH isozymes loci and (2) the LAP-1 and PGM-2 isozymes loci, which leads to the conclusion that the respective isozyme pairs are linked.In addition, a significant association (P value < 0.001) was observed between LAP-1 and GPI-2 when the pollen sources were Fritz, Mission, or Price, but this could not be tested for the remaining five pollen sources, Carmel, Grant, Keane, Ne plus Ultra, Peerless, because they are homozygous at these loci. If LAP-1 is linked with GPI-2 and PGM-2, it might be expected that we should find evidence of linkage between GPI-2 and PGM-2. The lack of a significant association between these two isozymes suggests that LAP-1 is located centrally on the chromosome. These three pairs of linked loci are the first to be reported in almond.     

The response of a cytokinin resistant mutant is highly specific and permits a new cytokinin bioassay

We have previously isolated six independent cytokinin-resistant mutants of Nicotiana plumbaginifolia which define three complementation groups, zeal, zea2 and zea3. We report here the characterization of the phenotypic response to cytokinin treatment of the mutant 1–64, belonging to the zeal group, and the result of the study of the specificity of this response. The phenotype of this mutant grown in the presence of cytokinin concentrations higher than 0.1 M is characterized by a hypertrophy of the cotyledons and hypocotyl which results in an increase of plantlet fresh weight. This hypertrophy is correlated to cytokinin concentration in a range between 0.01 to 10 M. The specificity of this response has been verified by using adenine and urea type cytokinins, as well as enantiomers of methylzeatin and methylbenzyladenine which differ widely in their cytokinin activities. We show that the high specificity of the hypertrophic response to cytokinins can be used as a convenient bioassay to screen the cytokinin activity of adenine or urea type molecules.Abbreviations zeatin [6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine] - iP isopentenyladenine [6-(3-methylbut-2-enylamino)purine] - BA benzyladenine [6-(benzylamino)purine] - (R)-(+)-MeZea [(R)--methylzeatin] - (S)-(–)-MeZea [(S)--methylzeatin] - (R)-(+)-MeBA [(R)--methylbenzyladenine] - (S)-(–)-MeBA [(S)--methylbenzyladenine] - CPPU N-(2-chloro-4-pyridyl)-N-phenylurea - thidiazuron N-(1,2,3-thiadiazol-5-pyridyl)-N-phenylurea The authors dedicate this paper to the memory of Jean-Pierre Bourgin, Director of the Laboratoire de Biologie Cellulaire, who died suddenly on October 29, 1994.     

Ingestion of the cyanobacterium Trichodesmium spp. by pelagic harpacticoid copepods Macrosetella,Miracia and Oculosetella

Trichodesmium is a filamentous, colonial nitrogen fixing cyanobacteria, ubiquitous in tropical and subtropical regions of the world's oceans. Trichodesmium fixes atmospheric nitrogen and can comprise a significant fraction of total primary production in oceanic surface waters. Therefore, the consumption and fate of Trichodesmium has important consequences for understanding carbon and nitrogen cycling in the open ocean. The pelagic harpacticoid copepod Macrosetella gracilis uses Trichodesmium not only as a physical substrate for juvenile development, but also as a food source. Several different types of pelagic copepods (including several species of calanoids, harpacticoids and a poecilostomatoid species) were tested for ingestion of Trichodesmium by labelling the cyanobacteria with ¹⁴C. Only the pelagic harpacticoids ingested Trichodesmium. Here we report the first grazing rates based on ¹⁴C-uptake measurements for Macrosetella gracilis (0.173 µg C copepod^–1 h^–1), and the first quantitative measurements of both Miracia efferata (0.402 µg C copepod^–1 h^–1) and Oculosetella gracilis (0.126 µg C copepod^–1 h^–1) ingesting this cyanobacteria. Ingestion rates of M. gracilis and M. efferata on the two different species of Trichodesmium, T. thiebautii and T. erythraeum, as well as the two different colonial morphologies of T. thiebautii, spherical-shaped (puffs) and fusiform (tufts), were also compared. Both Miracia and Macrosetella had higher ingestion rates on the puff colonies than the tuft colonies of T. thiebautii.. Both also had higher ingestion rates of T. erythraeum than T. thiebautii. Trichodesmium thiebautii contains a previously reported neurotoxin which may be an important factor in determining trophodynamic interactions. Our results suggest that pelagic harpacticoid copepods can be quantitatively important in determining the fate of Trichodesmium carbon and nitrogen.     

Satellited chromosomes,systematics and phylogeny inTaraxacum (Asteraceae)

A survey is made of the occurrence, nature and frequency of satellited chromosomes in the agamospermous genusTaraxacum. Species belonging to the 10 sections thought to be most primitive in the genus lack satellited chromosomes. In most other sections, a characteristic satellited chromosome is seen with a large euchromatic region distal to the presumed nucleolar oraniser region (NOR). In sections of a precursor type, there is always one chromosome of this Taraxacum type per haploid genome. In sections thought to be of an advanced type the number of such satellited chromosomes is very unstable, sometimes even within the same tissue. In sectionHamata, two such satellited chromosomes are invariably found in triploids. This finding strongly supports the integrity of this section, suggests that the species of the section are monophyletic, and have evolved from a single ancestor subsequent to the occurrence of obligate agamospermy. In three sections of the genus, satellited chromosomes of the conventional type with a very small distal euchromatic region distal to the NOR are reported for the first time in the genus.     

A simple method for maintaining high multiplication of Narcissus shoot cultures in vitro

A simple method for stimulating and maintaining high in vitro multiplication of Narcissus shoot clump cultures was developed. Shoot clumps were subjected either to normal cutting where leaves were trimmed to 20 mm in length at the beginning of each culture passage or to severe cutting where shoot clumps were cut down to the basal plate region removing all green tissue. Severe cutting at the beginning of each culture passage initially doubled the leaf multiplication, compared to normal cutting, but the difference between cutting treatments declined in successive passages. The improvement in leaf multiplication was maintained when shoot clumps were subjected to severe cutting only at every other culture passage, with no cutting in the alternate recovery passages. In vitro multiplication was increased by severe cutting in all seven Narcissus cultivars which were tested.Abbreviations NAA-1 naphthylacetic acid - BAP benzylaminopurine     

Functional expression of keratinase (kerA) gene from Bacillus licheniformis in Pichia pastoris

A 1.2 kb DNA fragment coding for the pro-peptide and mature keratinase from Bacillus licheniformis PWD-1 (kerA) was cloned into vectors pPICZA and pGAPZA for extracellular expression in the methylotrophic yeast, Pichia pastoris. Recombinant keratinase was secreted by the pPICZA-kerA transformants 24 h after methanol induction of shake-flask cultures, and reached a final yield of 124 mg l^–1 (285 U ml^–1) 144 h after the induction. The recombinant keratinase was glycosylated ( 39 kDa), and was optimal between pH 8.5–9.5 and between 55°C –60°C using azokeratin as substrate. The enzyme degraded bovine serum albumin, collagen, and soy protein concentrate. In conclusion, P. pastoris can be used as an efficient host to express keratinase for nutritional and environmental applications.     

Effects of the potential allelochemical α-asarone on growth,physiology and ultrastructure of two unicellular green algae

The effects of the natural phenylpropanoid -asarone on growth pattern, photosynthesis, respiration and cell structure of two microalgae have been investigated. In cultures ofAnkistrodesmus braunii -asarone decreases in the medium and induces a lag in growth. Both phenomena were dependent on the number of cells inoculated. By contrast, in cultures ofSelenastrum capricornutum a constant decrease of the growth rate at all inocula was observed and only a slight decrease of -asarone in the medium occurred. In both algae -asarone caused an initial inhibition of photosynthesis, followed by a resumption of control values. The respiratory rate ofA. braunii was not significantly affected by -asarone, whereas inS. capricornutum respiration lowered to 60% of the control in the first 48 h and subsequently rose to values exceeding the controls by 20%. Ultrastructural observations carried out 24 and 72 h after the addition of -asarone showed modifications of cell wall inA. braunii, an increase in the number of mithocondrial profiles per cell section inS. capricornutum, and an accumulation of electron-dense deposits in the vacuoles of both algae.Author for correspondence     

The carbohydrate structure of human fibronectins: A comparison between normal embryonic lung fibroblasts WI-38 and the SV40 virus transformed cell line VA13

The carbohydrates of human fibronectin released from non-transformed human fibroblasts WI-38 have been compared with those of fibronectin released from SV40 virus transformed WI-38/VA13 cells and those of fibronectin prepared from human plasma. The majority of the bi-antennary glycopeptides of fibronectin released from WI-38 fibroblasts was not sialylated at the terminal galactosyl residues, but was fucosylated at the coreN-acetylglucosaminyl residue directly linked to a peptide (structure A, below). Most of the minor sialylation detected was linked 2–3 to galactose. In contrast, the majority of the bi-antennary glycopeptides released from the transformed VA13 cells was highly sialylated at the terminal galactosyl residues with both 2–3 and 2–6 linkages, but was only partially fucosylated at the coreN-acetylglucosaminyl residue (structure B, below). This structure was similar to that of the bi-antennary glycopeptide of human plasma fibronectin which was, however, predominantly sialylated with an 2–6 linkage (structure C, below). These human fibronectins, regardless of their source, lack a high molecular weight lactosaminoglycan structure.In addition to the differences in bi-antennary structure described above, the quantity of tri- to tetra-antennary glycopeptides of fibronectin released from VA13 transformed cells was found to be much greater than the quantity of these glycopeptides of fibronectin released from normal WI-38 fibroblasts. Furthermore, there was a relatively small quantity of a glycopeptide fraction having a smaller molecular weight that did not bind to Con A-Sepharose and was separated on a Bio-Gel P-4 column. The presence of this fraction was characteristic for fibronectin released from transformed VA13 cells, and the fraction was absent in fibronectin from normal fibroblasts.     

Polysaccharide-enriched fraction isolated from Duchesnea chrysantha protects against oxidative damage

Reactive oxygen species (ROS)-related oxidative damages have been implicated in a wide variety of the pathological processes such as atherosclerosis, inflammation and carcinogenesis. A polysaccharide-enriched fraction (PEF) was isolated from Duchesnea chrysantha, a herbaceous plant, and its antioxidant activity was demonstrated using several assay systems in vitro. The PEF effectively inhibited Cu²⁺-stimulated low density lipoprotein oxidation in a concentration-dependent way and retarded the conjugated diene formation with the enhancement of the lag phase during oxidation. An assay for DNA strand breaks showed that PEF strongly protected DNA against damage caused by UV or by OH or O₂ ^– generated in the metal-catalyzed oxidation system. PEF effectively inhibited Nitroblue Tetrazolium reduction mediated by O₂ ^– in a dose-dependent manner. It also competed with 2-deoxy-d-ribose in absorbing OH generated by -irradiation (600 Gy) and thus inhibited the formation malondialdehyde. In conclusion, these evidences indicate that PEF may act as an antioxidant to scavenge O₂ ^–, OH or LO₂ directly. Our findings suggest the possibility that it may play a role as a potential therapeutic antioxidant in treatment of oxidative damage-derived diseases.     

Benzyl-N-acetyl-α-d-galactosaminide inhibits the sialylation and the secretion of mucins by a mucin secreting HT-29 cell subpopulation

We have analysed the mucins synthesized by the HT-29 MTX cell subpopulation, derived from the HT-29 human colon carcinoma cells through a selective pressure with methotrexate (Lesuffleuret al., 1990,Cancer Res 50: 6334–43), in the presence of benzyl-N-acetyl--galactosaminide (GalNAc-O-benzyl), which is a potential competitive inhibitor of the 1,3-galactosyltransferase that synthesizes the T-antigen. The main observation was a 13-fold decrease in the sialic acid content of mucins after 24 h of exposure to 5mm GalNAc-O-benzyl. This effect was accompanied by an increased reactivity of these mucins to peanut lectin, testifying to the higher amount of T-antigen. The second observation was a decrease in the secretion of the mucins by GalNAc-O-benzyl treated cells. The decrease in mucin sialyation was achieved through thein situ -galactosylation of GalNAc-O-benzyl into Gal1–3GalNAc-O-benzyl, which acts as a competitive substrate of Gal1–3GalNAc 2,3-sialyltransferase, as shown by the intracellular accumulation of NeuAc2–3Gal1–3GalNAc-O-benzyl in treated cells.Abbreviations BSM bovine submaxillary mucin - MTX methotrexate - PBS sodium phosphate 10mm, NaCl 0.15m, pH 7.4 buffer - pNp p-nitrophenol - TBS Tris/HCl 10mm, NaCl 0.15m, pH 7.4 buffer Enzymes: CMP-NeuAc: Gal1–3/4GlcNAc 2,3-sialyltransferase, ST3(N), EC 2.4.99.6; CMP-NeuAc: Gal1–4GlcNAc 2,6-sialyltransferase, ST6(N), EC 2.4.99.1; CMP-NeuAc: Gal1–3GalNAc 2,3-sialyltransferase, ST3(O), EC 2.4.99.4; CMP-NeuAc: R-GalNAc1-O-Ser 2,6-sialyltransferase, ST6(O)-I, EC 2.4.99.3; CMP-NeuAc: NeuAc2–3Gal1–3GalNAc 2,6-sialyltransferase, ST6(O)-II, EC 2.4.99.7; UDP-GlcNAc: Gal1–3GalNAc-R·(GlcNAc to GalNAc) 1,6-N-acetylglucosaminyltransferase, EC 2.4.1.102; UDP-GlcNAc: GalNAc-R 1,3-N-acetylglucosaminyltransferase, EC 2.4.1.147; UDP-Gal: GalNAc-R 1,3-galactosyltransferase, EC 2.4.1.122.     

The farmed Eucheuma species (Gigartinales,Rhodophyta) in Danajon Reef,Philippines: carrageenan properties

Six cultured strains of Eucheuma denticulatum and E. alvarezii, from which stocks can be selected for the development of a Eucheuma seedling bank, were tested for their carrageenan quality from June to November 1988. Percent yield of all the varieties taken together was apparently higher in June, becoming lower in November (regression, r –0.785, probability, p 0.001). Stepwise regression analysis was done to determine the existence of any relationship between any of the following parameters: gel strength, viscosity, sulfate content, month of sampling, and yield, whether taken individually or in combination. Results show variations of the yield with the month of sampling. ANOVA was performed to test whether there are differences in sulfate levels, gel strength, and viscosity between the Eucheuma alvarezii morphotypes. There was no significant difference between the green and the brown types.     

MucAB but not UmuDC proteins enhance ?2 frameshift mutagenesis induced by N-2-acetylaminofluorene at alternating GC sequences

N-2-acetylaminofluorene has been shown efficiently to induce both –1 and –2 frameshift mutations in Escherichia coli as well as in mammalian cells. In E. coli, the genetic characteristics of –1 and –2 frameshift mutations were found to be distinct. The –1 frameshift mutation pathway occurs at monotonous runs of G residues (i.e. GGGGG). This pathway exhibits the same genetic requirements as UV light-induced base substitution mutagenesis. Indeed, optimal mutagenesis requires the expression of both UmuDC and the activated form of RecA. The –2 frameshift mutation pathway operates at short alternating GpC sequences, such as the NarI sequence (i.e. GGCGCCGGCC). In contrast to the –1 frameshift mutation pathway, optimal induction does not require the UmuDC and RecA proteins. This pathway involves a LexA-repressed function tentatively called Npf (for NarI processing factor). In this paper, we show that MucAB efficiently stimulates the –2 frameshift mutation pathway. However, unlike the Npf pathway, MucAB-mediated stimulation requires expression of the RecA protein.     

Analysis of regions essential for the function of chromosomal replicator sequences from Yarrowia lipolytica

Summary Two DNA segments exhibiting ARS (autonomously replicating sequence) activity in the dimorphic yeast Yarrowia lipolytica were cloned from its chromosome on an integrative LEU2 plasmid. These ARS segments, designated YlARS1 and YlARS2, conferred on the hybrid plasmids high transformation efficiency and enabled extrachromosomal transmission of the plasmids in 1 or 2 copies per yeast cell under selective conditions. Deletion analysis showed that at least 728–1003 by for YlARS1 and 1377–1629 by for YlARS2 were required for full function. Both of these regions contained two 10/11 matches to an ARS core consensus in Saccharomyces cerevisiae, whereas neither was similar to the S. cerevisiae centromere sequence. Significantly, both YlARS elements contained at, or close to, their boundaries a 13 bp sequence, 5-TATATTCAAGCAA-3, which resembles the cleavage site for topoisomerase II in Drosophila. A central 524 by ClaI fragment of YlARS2 contained four stretches of a 17 bp direct repeat sequence, 5-GAAAAACAAAAACAGGC-3, and exhibited the electrophoretic behavior typical of bent DNA.