bluuebunny/crossref_metadata_2025_split huggingface dataset:
The duplex DNA is very underwound in the three‐stranded RecA protein‐mediated synaptic complex
Authors: Voloshin Oleg N. , Camerini‐Otero R. Daniel 
Published: November 2003

Background:The RecA protein is a central player in bacterial homologous recombination. It promotes two key events: the search for homology between two DNA molecules and the subsequent formation of the synaptic complex composed of RecA and three DNA strands (two from one duplex and one single strand). In spite of numerous studies, the architecture of the synaptic complex is still far from clear.Results:We have exploited two approaches to study the structure of Escherichia coli RecA protein‐mediated DNA synapsis: chemical modification with potassium permanganate and treatment of synaptic complexes of different lengths with topoisomerase I. The linking number difference values, obtained after separation of the individual sets of topoisomers in an agarose gel, were used to determine the number of bases per helical turn. We were able to show that the topology of the three‐stranded complexes containing RecA is quite different from that expected for deproteinized D‐loops. The original duplex in the synaptic complex is unwound, but not necessarily unpaired, to a structure topologically equivalent to DNA with approximately 27 bp per turn. Despite the fact that the patterns of reactivity towards potassium permanganate cannot be interpreted unambiguously, the results of chemical footprinting can be explained in terms of a synaptic complex as an extended and unwound three‐stranded helical structure.Conclusions:This work provides the first quantitative topological parameters for the RecA protein‐mediated three‐stranded synaptic complex. Furthermore, the structure of synaptic complexes is different from that of a simple D‐loop.
A complete Not I restriction map covering the entire long arm of human chromosome 11
Authors: Hosoda Fumie , Arai Yasuhito , Kitamura Eiko , Inazawa Johji , Fukushima Michiyo , Tokino Takashi , Nakamura Yusuke , Jones Carol , Kakazu Naoki , Abe Tatsuo , Ohki Misao 
Published: November 2003

Background:Human chromosome 11 is one of the autosomes on which many disease genes have been mapped. Many different types of map, including a radiation hybrid map, a genetic map, and an STS‐content YAC map, have been constructed for the chromosome. However, a physical map providing accurate physical distances has not yet been established. A chromosome‐wide Not I restriction map was constructed to understand the overall feature of the genome organization and to facilitate the positional cloning of disease genes.Results:A complete Not I restriction map of the entire long arm of human chromosome 11 was constructed using linking‐clone mapping. This physical map covers 77.6 Mb, from a pericentromeric Not I site to the terminus, and provides the most accurate ordering and distance estimation to date. We also mapped 138 sequence markers in the q13 region that have been poorly mapped previously.Conclusions:The restriction map of the entire long arm of human chromosome 11 is the longest restriction map of the human genome. This mapping has disclosed unique features regarding the organization of the chromosome, indicating that restriction sites of Not I, a CpG‐recognition enzyme, are primarily distributed in R (or T) bands and that genetic distance is considerably longer in R (or T) bands than in G bands. The mapping, as well as the dense concentration of mapped markers within the q13 region, should help with positional cloning of the genes associated with various diseases.
Two mechanisms in the action of repressor δEF1: binding site competition with an activator and active repression
Authors: Sekido Ryohei , Murai Kasumi , Kamachi Yusuke , Kondoh Hisato 
Published: November 2003

Background:Counteraction between activators and repressors is crucial for the regulation of a number of cell‐specific enhancers, where an activator and a repressor are mutually competitive in binding to the same site. δEF1 is a repressor protein of δ1‐crystallin minimal enhancer DC5 binding at the CACCT site, and inhibits activator δEF3 from binding to the overlapped site. It has two zinc finger clusters N‐fin and C‐fin, close to N‐ and C‐termini, respectively, and a homeodomain in the middle. δEF1 also binds to the E2‐box sequence CACCTG, and represses E2‐box‐dependent enhancers.Results:The mechanism of the repressor action of δEF1 was investigated by examining various deletion mutants of δEF1 for their activity to repress δ1‐crystallin enhancer fragment HN which contained DC5 sequence and an additional activator site. Both zinc finger clusters were found to be essential for DNA binding and repression, but the homeodomain was not. In addition, the NR domain close to the N‐terminus was required for full repression. The NR domain showed active repression when fused to the Gal4 DNA binding domain. Active repression by δEF1, dependent on the NR domain, was also demonstrated in a situation where the binding sites of δEF1 and δEF3 were separated.  N‐fin and C‐fin in their isolated forms bind the 5′‐(T/C)ACCTG‐3′ and 5′‐(t/C)ACCT‐3′ sequences, respectively, while the homeodomain showed no DNA binding activity. An analysis of DNA binding of the ΔIntF form, having both N‐fin and C‐fin, indicated that a single DNA binding domain is assembled from two zinc finger clusters.Conclusion:Two mechanisms are involved in the repressor action of δEF1. First, a binding site competition with an activator which depends on the integrity of both zinc finger clusters, and second, an active repression to silence an enhancer which is attributed to the NR domain.
Roles of TraI protein with activities of cleaving and rejoining the single‐stranded DNA in both initiation and termination of conjugal DNA transfer
Authors: Fukuda Hirokazu , Ohtsubo Eiichi 
Published: November 2003

Background:The plasmid R100 encodes the TraI protein, which is required for conjugal DNA transfer. TraI has the activity of site‐ and strand‐specific nicking of the supercoiled plasmid DNA. The molecular mechanism of this specific nicking, which is supposed to be the initiation reaction of DNA transfer, is not understood.Results:We have demonstrated that TraI has the ability to cleave the single‐stranded DNA at the same site as the nicking site (nic) in a region, which we here refer to as sbi. The product contained the TraI protein which was covalently linked to the newly generated 5′ end of the nicking reaction. Both the cleaving and nicking reactions took place under almost the same conditions and required the presence of the sbi region. DNase I‐footprinting analysis revealed that the TraI bound to the single‐stranded DNA of the sbi region. TraI did not cleave the double‐stranded DNA fragment, but it did cleave the double‐stranded DNA with a single‐stranded DNA portion in the sbi region. KMnO4 mapping analysis revealed that TraI can melt the sbi region in the supercoiled DNA to generate a single‐stranded portion. We have also demonstrated that TraI was able to rejoin the cleaved products. The rejoining reaction required the 5′ end of one cleaved product with the TraI covalently attached and the 3′ end of the other product containing the sbi region.Conclusions:Our results demonstrate that the nicking reaction—the initiation reaction of DNA transfer—is actually the cleaving reaction of the single‐stranded DNA. TraI, which has both cleaving and rejoining activities, is thought to be involved in the termination of DNA transfer, to give a copy of the conjugative plasmid by joining the 5′ end, which is generated by the initiation reaction, with the 3′ end, which will be generated upon cleavage of the sbi region appearing after one round of the rolling circle replication of the plasmid.
Regions of the Escherichia coli primary sigma factor σ70 that are involved in interaction with RNA polymerase core enzyme
Authors: Nagai Hiroki , Shimamoto Nobuo 
Published: November 2003

Background:The σ factors of bacterial RNA polymerase are required for recognition of promoters in transcription initiation. Most σ factors share several regions with significant homology in their amino acid sequences (regions 1–4). Some primary σ factors carry a large nonconserved segment between regions 1 and 2. The binding of an σ factor to the core enzyme alters the structure and properties of the σ factor, but little is known about the binding mechanism and subsequent reactions. In this report, we employed the protein footprinting method to investigate the alteration of the structure and function of Escherichia coli σ70 by binding to core enzyme and promoter DNA.Results:A segment between regions 1.1 and 1.2, and that in region 3.2, were preferentially cleaved by hydroxyl radicals. Upon binding to the core enzyme, segments in regions 1.1, 2, 3 and 4 were substantially protected, while cleavage at a small segment in region 4.2 was weakly enhanced. In a binary complex of holoenzyme and promoter DNA, additional segments in regions 2.4 and 4.2 were protected, while the protection at region 1.1 disappeared. The nonconserved acidic region of σ70 in the holoenzyme became hypersensitive upon binding to DNA.Conclusions:These results suggest that not only the conserved region 2, but also regions 1.1, 3 and 4 of the σ factor are involved in binding to the core enzyme. The nonconserved acidic region is likely to be more exposed by further binding of σ factor to promoter DNA.
Role of the Drosophila EGF receptor in determination of the dorsoventral domains of escargot expression during primary neurogenesis
Authors: Yagi Yoshimasa , Hayashi Shigeo 
Published: November 2003

Background
: Primary neurogenesis in the central nervous system of insects and vertebrates occurs in three dorsoventral domains in each side of the neuroectoderm. Among the three dorsoventral domains of the Drosophila neuroectoderm, the medial and lateral columns express the zinc‐finger gene escargot (esg), whereas the intermediate column does not. We studied esg expression as a probe to investigate the mechanism of neuroectoderm patterning.
Results
: The effect of dorsoventral patterning genes on esg expression was studied. decapentaplegic, snail and twist were found to repress esg expression outside the neuroectoderm. The expression of esg in the intermediate column is normally repressed, but was de‐repressed when the EGF receptor homologue (DER) activity was either elevated or reduced. A neurogenic enhancer of esg was identified, and was shown to be separable into a distal region that promotes ubiquitous expression in the neuroectoderm and a proximal region that represses the intermediate expression.
Conclusions
: decapentaplegic, snail, twist and an activator act through the distal region to initiate transcription of esg in the neuroectoderm. We propose that the combination of opposing gradients of DER and its ligand creates a peak of DER activity in the intermediate column where DER represses esg transcription through the proximal repressor region. These two kinds of regulation establish the early esg expression that prefigures the neuroectoderm patterning.
Involvement of the MRE2 gene of yeast in formation of meiosis‐specific double‐strand breaks and crossover recombination through RNA splicing
Authors: Nakagawa Takuro , Ogawa Hideyuki 
Published: November 2003

Background
: The mre2 mutant of Saccharomyces cerevisiae is defective in meiotic recombination and produces inviable spores, but the sensitivities to DNA damaging agents, methyl methanesulphonate and ultraviolet light are not altered by the mutation. Mre2 has two copies of RNA recognition motif (RRM), suggesting its participation in RNA metabolism in meiosis.
Results
: An amino acid substitution in the N‐terminal RRM of Mre2 confers a meiotic recombination defect. Using this mre2N strain, the MER2 gene was isolated as a multi‐copy suppressor of the recombination defect. Meiosis‐specific splicing of MER2 pre‐mRNA was impaired in the mre2 deletion (mre2Δ) mutant. The mre2Δ mutant was defective in the formation of meiosis‐specific double‐strand breaks (DSBs) and crossover and noncrossover recombinants. When the chromosomal MER2 gene was replaced with the intronless derivative of MER2 gene, cMER2, the formation of DSBs and of noncrossover recombinants were restored in the mre2Δ mutant. However, the amount of crossover recombinants produced in the mre2ΦcMER2 strain was ≈30% that in the wild‐type. In addition, the mre2ΔcMER2 mutant was defective in chromosome segregation and in viable spore formation.
Conclusions
: Mre2 participates in the formation of DSBs through meiosis‐specific splicing of MER2 pre‐mRNA. Besides, Mre2 is also involved in crossover recombination, possibly through splicing of RNA from another gene(s).
Phenotypic analysis of null mutants for DE‐cadherin and Armadillo in Drosophila ovaries reveals distinct aspects of their functions in cell adhesion and cytoskeletal organization
Authors: Oda Hiroki , Uemura Tadashi , Takeichi Masatoshi 
Published: November 2003

Background
: DE‐cadherin is an epithelial cadherin in Drosophila, and forms adherens junctions by associating with Armadillo (β‐catenin). To investigate its role in oogenesis, we generated germ‐line clones homozygous for a null mutation in shotgun (shg) encoding this molecule, and examined their phenotypes, comparing with those of armadillo (arm) mutants.
Results
: In the wild‐type ovaries, DE‐cadherin was expressed by both the germ‐line and somatic derivatives, colocalizing with Armadillo. In the shg mutant ovaries in which the mutation was restricted to the germ line, germ cells were rounded, and generated gaps between themselves, suggesting that their surface adhesiveness was reduced or lost. However, the positioning of germ cells in the egg chamber was normal. Two groups of somatic follicle cells—the border cells and centripetal follicle cells—frequently migrated along incorrect pathways, indicating that DE‐cadherin is required for their appropriate migration. Notably, the shg phenotypes were distinct from those of arm null mutants. Intercellular adhesion appeared to be less severely affected by arm than by the shg mutation, and the actin‐based cytoskeleton and cell arrangement were disorganized only in the arm mutants.
Conclusions
: These findings suggest that DE‐cadherin is critical for cell–cell adhesion, and functional to a certain extent without Armadillo, whereas Armadillo is required for cytoskeletal organization and for the control of cell positioning. We therefore propose that the molecular complex of DE‐cadherin and Armadillo which is present in normal cells is endowed with multiple functions derived from each molecule.
Molecular properties of the proteasome activator PA28 family proteins and γ‐interferon regulation
Authors: Tanahashi Nobuyuki , Yokota Kin‐ya , Ahn Joon Young , Chung Chin Ha , Fujiwara Tsutomu , Takahashi Ei‐ichi , DeMartino George N. , Slaughter Clive A. , Toyonaga Tetsushi , Yamamura Ken‐ichi , Shimbara Naoki , Tanaka Keiji 
Published: November 2003

Background: Recent cDNA cloning of two homologous proteasome activators, PA28α and PA28β, indicated the presence of a structurally related third protein, Ki antigen, but a functional relationship between Ki antigen and the two PA28 proteins is unknown. Accumulating evidence has implicated an important role for PA28 in the major histocompatibility complex (MHC) class I‐restricted antigen processing pathway. Recently, an immunomodulatory cytokine γ‐interferon (γ‐IFN) was found to increase greatly the messages for PA28α and PA28β, but not Ki antigen, in human cells. Results: Ki antigen was co‐immunoprecipitated with the 20S proteasome by anti‐proteasome antibody, and associated reversibly with the 20S proteasome, as observed for PA28α and PA28β. Therefore, Ki antigen was renamed PA28γ. Anti‐PA28γ antibody, however, did not immunoprecipitate PA28α and PA28β. γ‐IFN caused an almost complete loss of the PA28γ protein in cells without affecting its mRNA level, whereas the levels of both mRNA and protein for PA28α and PA28β were coordinately up‐regulated by γ‐IFN. Finally we showed that the human chromosomal genes of PA28α and PA28γ were located on 14q11.2 and 17q21.32‐21.33, respectively. Conclusion: PA28γ (equivalent to Ki antigen) is a new member of the PA28 family proteins. It exists as a unique homopolymer under non‐denaturing conditions. γ‐IFN was found to induce the expression of PA28α and PA28β, whereas it caused almost complete loss of the PA28γ protein in cells. The reciprocal expression of the PA28 family proteins may imply their involvement in distinct biological processes.
Basal level transcription of the human hsp86 gene is directed by intron‐based elements
Authors: Lange Peer , Victor Martin , Benecke Bernd‐Joachim 
Published: November 2003

Background: The heat shock response includes the syntheses of three major size‐classes of heat shock proteins (hsps) designated as class 20, 70 and 90 hsps, respectively. In contrast to the class 20 and 70 hsp genes, those coding for the class 90 hsps are already actively transcribed in the non‐induced state. Results: We have shown that the increased basal level transcription of the human hsp86 gene depends on the presence of the first intron. Furthermore, intron I of the hsp86 gene was also capable of conferring increased basal level transcription on the human hsp70 promoter. Finally, the intron I‐based sequence elements of the hsp86 gene mediated an effective rescue of 5′‐truncated hsp70 and hsp86 promoters. In contrast, such a rescue of inactivated promoters was not obtained with the viral SV40 gene enhancer which is often also capable of stimulating basal level expression. Conclusions: Element(s) which direct the basal transcription of the human hsp86 gene at normal physiological temperatures are located within the first intron of the gene. It is conceivable that this intron‐dependent activation of basal hsp86 transcription reflects a fundamental requirement for this protein in the context of cellular growth and metabolism under non‐stress conditions.
atonal is a proneural gene for a subset of olfactory sense organs in Drosophila
Authors: Gupta Bhagwati Prasad , Rodrigues Veronica 
Published: November 2003

Background: The antenna of the adult fruit fly, Drosophila melanogaster, is covered with three morphologically distinct types of olfactory sense organs. In addition, mechano‐ and hygro‐sensitive receptors are also present on its surface. While much has been learnt about the development of peripheral nervous system in Drosophila, the mechanisms underlying the development of olfactory sensilla are just beginning to be unraveled. The antennal sense organs have several properties that make them distinct from other sense organs. While each sensillum type is arranged in a well‐defined region of the antenna, the position of an individual sensillum is not fixed. The development of these sense organs appears to combine an initial step of cell recruitment, as in photoreceptors, followed by cell lineage mechanisms, as in the development of other external sense organs. The earliest step in development, the selection of a sensory organ precursor, involves the interaction of proneural and neurogenic genes. The proneural gene for the antennal sense organs has been elusive so far. Results: We show that the basic helix‐loop‐helix (bHLH) transcription factor encoded by atonal (ato) is a proneural gene for one morphological type of olfactory sensilla on the antenna and for all the olfactory sensilla on the maxillary palp. Loss of function and overexpression experiments together reveal that ato is both necessary and sufficient to specify these sensilla. Immunohistochemical experiments show that Ato expresses in a dynamic pattern in the developing antennal disc. Conclusions: Our results demonstrate that ato acts solely in the specification of antennal sensilla coeloconica. This along with our previous observation that the AS‐C genes do not function in antenna allows us to suggest that other proneural genes must operate in the specification of sensilla basiconica and trichoidea. Our experiment involving overexpression of extramacrochaetae, a negative regulator of bHLH encoding genes, results in a significant reduction in the number of all three types of antennal sensilla. This suggests that the unidentified proneural gene(s) possibly encode bHLH factors.
Topographic accessibility and the tectonic interpretation of gravity data
Authors: Daniel A. J. , Styles P. 
Published: November 2003

During recent gravity surveys in Patagonia, we were unable to acquire data on the North Patagonian Icecap, which is completely inaccessible for land surveying. The Icecap is clearly of tectonic significance, but, because of its tectonic history, it is uplifted and rugged, and consequently inaccessible. Therefore the distribution of geophysical observations which can be acquired in a rugged field area is dependent on the tectonic history. This suggests that a tectonic history which is reconstructed from geophysical data may be systematically biased.We use models of local and flexural isostasy to estimate the gravity errors associated with interpolation across inaccessible topography. The gravity error is largest for Pratt isostasy, where the mass deficit which supports the topography is at relatively shallow depths. The gravity error is least for flexural isostasy, because in this case the inaccessible topography is supported regionally by a mass deficit which extends beyond the inaccessible region.An analytical flexural interpolation scheme is proposed for interpolation across data gaps associated with inaccessible topography. Flexural theory and Gauss’s theorem are used to predict the gravity anomaly due to the mass excess of the inaccessible topography. We apply this scheme to the North Patagonian Icecap, to demonstrate that flexural interpolation predicts a relative gravity low at the site of the Icecap, which would not be predicted by purely geometrical interpolation schemes.
Elevated CO2 and moisture effects on soil carbon storage and cycling in temperate grasslands
Authors: TATE K.R. , ROSS D.J. 
Published: November 2003

In grassland ecosystems, most of the carbon (C) occurs below‐ground. Understanding changes in soil fluxes induced by elevated atmospheric CO2 is critical for balancing the global C budget and for managing grassland ecosystems sustainably. In this review, we use the results of short‐term (1–2 years) studies of below‐ground processes in grassland communities under elevated CO2 to assess future prospects for longer‐term increases in soil C storage.Results are broadly consistent with those from other plant communities and include: increases in below‐ground net primary productivity and an increase in soil C cycling rate, changes in soil faunal community, and generally no increase in soil C storage. Based on other experimental data, future C storage could be favoured in soils of moderate nutrient status, moderate‐to‐high clay content, and low (or moderateIy high) soil moisture status. Some support for these suggestions is provided by preliminary results from direct measurements of soil C concentrations near a New Zealand natural CO2‐venting spring, and by simulations of future changes in grassland soils under the combined effects of CO2 fertilization and regional climate change.Early detection of any increase in soil C storage appears unlikely in complex grassland communities because of (a) the difficulty of separating an elevated CO2 effect from the effects of soil factors including moisture status, (b) the high spatial variability of soil C and (c) the effects of global warming. Several research imperatives are identified for reducing the uncertainties in the effects of elevated atmospheric CO2 on soil C.