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A map of rat Chromosome (Chr) 10 was generated from 21 markers, mostly of conserved structural genes, by linkage analysis and fluorescence in situ hybridization. The study emphasizes the proximal third of the chromosome which, until now, has been relatively devoid of markers. Based on comparative analysis, our data suggest that genes on rat Chr 10 are conserved on mouse Chr 11, 16, 17 and human Chr 16, 5, and 17.
A retrospective records analysis of 32 patients with mandibular osteoradionecrosis seen between August 1960 and September 1995 was undertaken to determine the effectiveness of nonsurgical/nonhyperbaric oxygen conservative management. Two patients died before final analysis. Of those alive, 65.5% of patients were spared resection, having lesions resolve (48.3%), improve (3.4%), or asymptomatically stabilize (13.8%). Sequestrum production allowing "gentle" removal predicted a more favorable clinical course when managed conservatively compared with nonsequestrating lesions (p < 0.05). Criteria to define clinical osteoradionecrosis are proposed.
Nonstructural protein 3 (NS3) of hepatitis C virus (HCV) has two distinct domains, protease and helicase, that are essential for HCV proliferation. Therefore, NS3 is considered a target for anti-HCV treatment. To study RNA aptamers of the NS3 helicase domain, we carried out in vitro selection against the HCV NS3 helicase domain. RNA aptamers obtained after eight generations possessed 5' extended single-stranded regions and the conserved sequence (5'-GGA(U/C)GGAGCC-3') at stem-loop regions. Aptamer 5 showed strong inhibition of helicase activity in vitro. Deletion and mutagenesis analysis clarified that the conserved stem-loop is important and that the whole structure is needed for helicase inhibition. We compared the inhibition of helicase activity between aptamer 5 and 3'+-UTR of HCV.
Three regions in the Saccharomyces cerevisiae RNase P RNA have been identified, at positions Sce 87-94, Sce 309-316, and Sce 339-349, that contain nucleotides that are invariant in identity and position among all the known RNase P RNAs. To study the importance of these conserved RPR1 RNA regions in enzyme function, three independent mutational libraries were created in which the positions of invariant nucleotides were randomized simultaneously. Screening in vivo was used to identify viable RPR1 variants when reconstituted into holoenzyme in cells. Despite the universal evolutionary conservation, most of these positions tolerate certain sequence changes without severely affecting function. Most changes, however, produced subtle defects in cell growth and RNase P function, supporting the importance of these conserved regions. Isolation of conditional growth mutants allowed the characterization of the effects of mutations on cell growth, RPR1 RNA maturation, and activity of the holoenzyme in vitro. Kinetic analysis showed that viable variants were usually more defective in catalytic rate (Kcat) than in substrate recognition (Km).
OBJECTIVES: To evaluate the efficacy of conservative adjuvant therapy after revascularisation procedures in patients with peripheral arterial disease. DESIGN: meta-analysis. MATERIALS: English-language studies published from 1976 to 1997. METHODS: Reports on conservative therapies in patients with peripheral arterial disease after percutaneous transluminal angioplasty, endarterectomy, thromboendarterectomy or bypass grafting were eligible. Uncontrolled or retrospective studies, double reports or trials without clinical outcomes were excluded. Included studies were graded as level 1 (randomised and double- or assessor-blind), level 2 (open randomised), or level 3 (non-randomised comparative). (Loss of) patency, amputation, vascular events and mortality were the outcomes considered. When feasible, end-of-treatment results, either continuous or binary, were combined with appropriate statistical methods. RESULTS: Thirty-two studies were included. Compared to non-active control, aspirin with dipyridamole improved (loss of) patency (odds ratio (OR) 0.69, 95% confidence interval (CI), 0.53 to 0.90) and mortality (OR 0.80, 95% CI, 0.57 to 1.14); ticlopidine improved (loss of) patency (OR 0.53, 95% CI, 0.33 to 0.85) and amputation (OR 0.29, 95% CI, 0.08 to 1.01). Data on the effectiveness of vitamin-K inhibitors were not conclusive. CONCLUSIONS: Patients with peripheral arterial disease improve their outcome by receiving aspirin with dipyridamole or ticlopidine after a revascularisation procedure.
A new gene (POLL) encoding a novel DNA polymerase (Pol lambda) has been identified at mouse chromosome 19. Murine Pol lambda, consisting of 573 amino acid residues, has a 32% identity to Pol beta, involved in nuclear DNA repair in eukaryotic cells. It is interesting that Pol lambda contains all the critical residues involved in DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, that are conserved in Pol beta and other DNA polymerases belonging to family X. Murine Pol lambda, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity when assessed by in situ gel analysis. Pol lambda also conserves the critical residues of Pol beta required for its intrinsic deoxyribose phosphate lyase (dRPase) activity. The first 230 amino acid residues of Pol lambda, that have no counterpart in Pol beta, contain a BRCT domain, present in a variety of cell-cycle check-point control proteins responsive to DNA damage and proteins involved in DNA repair. Northern blotting, in situ hybridization analysis and immunostaining showed high levels of Pol lambda specifically expressed in testis, being developmentally regulated and mainly associated to pachytene spermatocytes. These first evidences, although indirect, suggest a potential role of Pol lambda in DNA repair synthesis associated with meiosis.
Fructose-1,6-(bis)phosphate aldolase is a ubiquitous enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-(bis)phosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceral-dehyde-3-phosphate or glyceraldehyde, respectively. Vertebrate aldolases exist as three isozymes with different tissue distributions and kinetics: aldolase A (muscle and red blood cell), aldolase B (liver, kidney, and small intestine), and aldolase C (brain and neuronal tissue). The structures of human aldolases A and B are known and herein we report the first structure of the human aldolase C, solved by X-ray crystallography at 3.0 A resolution. Structural differences between the isozymes were expected to account for isozyme-specific activity. However, the structures of isozymes A, B, and C are the same in their overall fold and active site structure. The subtle changes observed in active site residues Arg42, Lys146, and Arg303 are insufficient to completely account for the tissue-specific isozymic differences. Consequently, the structural analysis has been extended to the isozyme-specific residues (ISRs), those residues conserved among paralogs. A complete analysis of the ISRs in the context of this structure demonstrates that in several cases an amino acid residue that is conserved among aldolase C orthologs prevents an interaction that occurs in paralogs. In addition, the structure confirms the clustering of ISRs into discrete patches on the surface and reveals the existence in aldolase C of a patch of electronegative residues localized near the C terminus. Together, these structural changes highlight the differences required for the tissue and kinetic specificity among aldolase isozymes.
Gas composition of capillary blood was studied in 120 patients with laryngeal cancer, primarily of stage IIIa (T3N0M0). Hypoxia was present in all the examinees whether or not laryngostenosis was clinically apparent. The analysis has shown that conservative surgery of laryngeal cancer with application of tubal polymeric endoprostheses corrects ventilatory disorders and improves blood gas composition. The degree of correction of ventilatory disorders depends on the kind of respiratory intraoperative maintenance. Gas composition appeared significantly better in the group of patients exposed to transtracheal high-frequency artificial pulmonary ventilation than in the group exposed to volumic artificial pulmonary ventilation.
The ability to recognize and predict non-sigma54 promoters in the alphaproteobacteria is not well developed. In this study, 25 experimentally verified Sinorhizobium meliloti promoter sequences were compiled and used to predict the location of other related promoters in the S. meliloti genome. Fourteen candidate predictions were targeted for verification and of these at least 12 proved to be genuine promoters. As a result, the experimental identification of 12 novel promoters linked to genes rpoD, topA, rpmJ, trpS, ropB1, metC, rpsT, secE, trkH and three tRNA genes is reported. In all, 99 predicted and verified promoters are reported, including those linked with 13 tRNA genes, eight ribosomal protein genes and a number of other physiologically important or essential genes. On the basis of sequence conservation and a mutational analysis of promoter activity, the -35 and -10 consensus for these promoters is 5-CTTGAC-N17-CTATAT. This promoter structure, which seems to be widely conserved amongst several other genera in the alphaproteobacteria, shares significant similarity with, but is skewed by a 1 nt step from, the canonical Escherichia coli sigma70 promoter. Perhaps this difference is responsible for the observation that S. meliloti promoters are often poorly expressed in E. coli. In this regard, expression data from plasmid-borne gfp-reporter fusions to eight of the S. meliloti promoters verified in this work revealed that while these promoters were very active in S. meliloti and Agrobacterium tumefaciens only very low, near-background activity was detected in E. coli.
Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface. We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic Escherichia coli. Structural analysis indicated PapC folds as a beta-barrel with short extracellular loops and extensive periplasmic domains. Several periplasmic regions were localized, including two domains containing conserved cysteine pairs. Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding. The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo. Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal fim gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo. These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus.
Resources for biodiversity conservation are severely limited, requiring strategic investment. Understanding both the economic benefits and costs of conserving ecosystems will help to allocate scarce dollars most efficiently. However, although cost-benefit analyses are common in many areas of policy, they are not typically used in conservation planning. We conducted a spatial evaluation of the costs and benefits of conservation for a landscape in the Atlantic forests of Paraguay. We considered five ecosystem services (i.e., sustainable bushmeat harvest, sustainable timber harvest, bioprospecting for pharmaceutical products, existence value, and carbon storage in aboveground biomass) and compared them to estimates of the opportunity costs of conservation. We found a high degree of spatial variability in both costs and benefits over this relatively small (approximately 3,000 km(2)) landscape. Benefits exceeded costs in some areas, with carbon storage dominating the ecosystem service values and swamping opportunity costs. Other benefits associated with conservation were more modest and exceeded costs only in protected areas and indigenous reserves. We used this cost-benefit information to show that one potential corridor between two large forest patches had net benefits that were three times greater than two otherwise similar alternatives. Spatial cost-benefit analysis can powerfully inform conservation planning, even though the availability of relevant data may be limited, as was the case in our study area. It can help us understand the synergies between biodiversity conservation and economic development when the two are indeed aligned and to clearly understand the trade-offs when they are not.
Experimental investigations of eukaryotic enhancers suggest that multiple binding sites and trans-acting regulatory factors are often required for wild-type enhancer function. Genetic analysis of the stripe 2 enhancer of even-skipped (eve), an important developmental gene in Drosophila, provides support for this view. Given the importance of even-skipped expression in early Drosophila development, it might be predicted that many structural features of the stripe 2 enhancer will be evolutionarily conserved, including the DNA sequences of protein binding sites and the spacing between them. To test this hypothesis, we compared sequences of the stripe 2 enhancer between four species of Drosophila: D. melanogaster, D. yakuba, D. erecta and D. pseudoobscura. Our analysis revealed a large number of nucleotide substitutions in regulatory protein binding sites for bicoid, hunchback, Kruppel and giant, as well as a systematic change in the size of the enhancer. Some of the binding sites in D. melanogaster are either absent or modified in other species. One functionally important bicoid-binding site in D. melanogaster appears to be recently evolved. We, therefore, investigated possible functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation. This analysis revealed that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D. melanogaster embryos. Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native eve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein. We argue that stripe 2 eve expression in Drosophila evolution can be viewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe. We further hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly deleterious and adaptive mutations in regulatory protein binding sites as well as in the spacing between binding sites. This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer.
X-ray, phylogenetic and quantum chemical analysis of molecular interactions and conservation patterns of cis Watson-Crick (W.C.) A/G base-pairs in 16S rRNA, 23S rRNA and other molecules was carried out. In these base-pairs, the A and G nucleotides interact with their W.C. edges with glycosidic bonds oriented cis relative to each other. The base-pair is stabilised by two hydrogen bonds, the C1'-C1' distance is enlarged and the G(N2) amino group is left unpaired. Quantum chemical calculations show that, in the absence of other interactions, the unpaired amino group is substantially non-planar due to its partial sp(3) pyramidalization, while the whole base-pair is internally propeller twisted and very flexible. The unique molecular properties of the cis W.C. A/G base-pairs make them distinct from other base-pairs. They occur mostly at the ends of canonical helices, where they serve as interfaces between the helix and other motifs. The cis W.C. A/G base-pairs play crucial roles in natural RNA structures with salient sequence conservation patterns. The key contribution to conservation is provided by the unpaired G(N2) amino group that is involved in a wide range of tertiary and neighbor contacts in the crystal structures. Many of them are oriented out of the plane of the guanine base and utilize the partial sp(3) pyramidalization of the G(N2). There is a lack of A/G to G/A covariation, which, except for the G(N2) position, would be entirely isosteric. On the contrary, there is a rather frequent occurrence of G/A to G/U covariation, as the G/U wobble base-pair has an unpaired amino group in the same position as the cis W.C. G/A base-pair. The cis W.C. A/G base-pairs are not conserved when there is no tertiary or neighbor interaction. Obtaining the proper picture of the interactions and phylogenetic patterns of the cis W.C. A/G base-pairs requires a detailed analysis of the relation between the molecular structures and the energetics of interactions at a level of single H-bonds and contacts.
Prokaryote gene annotation is complicated by large numbers of short open reading frames (ORFs) that arise naturally from genetic code design. Historically, many hypothetical ORFs have been annotated as genes in microbes, usually with an arbitrary length threshold (e.g. greater than 100 codons). Given the use of such thresholds, what is the extent of genuine undiscovered short genes in the current sampling of prokaryote genomes? To assess rigorously the potential under-annotation of short ORFs with homology, we exhaustively compared the polyORFome--all possible ORFs in 64 prokaryotes (53 bacteria and 11 archaea) plus budding yeast--to itself and to all known proteins. The novelty of our analysis is that, firstly, sequence comparisons to/between both annotated and un-annotated ORFs are considered, and secondly a two-step disabled-homology filter is applied to set aside putative pseudogenes and spurious ORFs. We find that un-annotated homologous short ORFs (uhORFs) correspond to a small but non-negligible fraction of the annotated prokaryote proteomes (0.5-3.8%, depending on selection criteria). Moreover, the disabled-homology filter indicates that about a third of uhORFs correspond to putative pseudogenes or spurious ORFs. Our analysis shows that the use of annotation length thresholds is unnecessary, as there are manageable numbers of short ORF homologies conserved (without disablements) across microbial genomes. Data on uhORFs are available from http://pseudogene.org/polyo
A novel mammalian gene, Eif4g2, with a high degree of homology to the p82 subunit of the wheat germ eukaryotic translation initiation factor eIF-(iso)4F and mammalian eIF-4G has been isolated. Zoo blot analysis indicates that Eif4g2 is a single-copy gene that is highly conserved among vertebrates. Northern blot analysis shows that Eif4g2 is ubiquitously expressed at high levels in all human and mouse tissues examined. The 3810-nucleotide Eif4g2 cDNA contains a 907-amino-acid open reading frame that codes for a polypeptide with a predicted molecular mass of 102 kDa. The Eif4g2 polypeptide exhibits an overall similarity to wheat p82 of 52%. A 248-amino-acid segment at the amino-terminal end of both peptides exhibits 63% similarity and contains conserved potential RNA binding domains and a phosphorylation site. The Eif4g2 polypeptide contains multiple potential N-linked glycosylation sites as well as protein kinase C and casein kinase II phosphorylation sites. Southern blot analysis of DNA from interspecific backcross mice shows that Eif4g2 is localized to distal mouse chromosome 7 in a region syntenic with human chromosome 11p15.
We describe the molecular analysis of the dpy-20 gene in Caenorhabditis elegans. Isolation of genomic sequences was facilitated by the availability of a mutation that resulted from insertion of a Tc1 transposable element into the dpy-20 gene. The Tc1 insertion site in the m474::Tc1 allele was identified and was found to lie within the coding region of dpy-20. Three revertants (two wild-type and one partial revertant) resulted from the excision of this Tc1 element. Genomic dpy-20 clones' were isolated from a library of wild-type DNA and were found to lie just to the left of the unc-22 locus on the physical map, compatible with the position of dpy-20 on the genetic map. Cosmid DNA containing the dpy-20 gene was successfully used to rescue the mutant phenotype of animals homozygous for another dpy-20 allele, e1282ts. Sequence analysis of the putative dpy-20 homologue in Caenorhabditis briggsae was performed to confirm identification of the coding regions of the C. elegans gene and to identify conserved regulatory regions. Sequence analysis of dpy-20 revealed that it was not similar to other genes encoding known cuticle components such as collagen or cuticulin. The dpy-20 gene product, therefore, identifies a previously unknown type of protein that may be directly or indirectly involved in cuticle function. Northern blot analysis showed that dpy-20 is expressed predominantly in the second larval stage and that the mRNA is not at all abundant. Data from temperature shift studies using the temperature-sensitive allele e1282ts showed that the sensitive period also occurs at approximately the second larval stage. Therefore, expression of dpy-20 mRNA and function of the DPY-20 protein are closely linked temporally.
The two mechanisms for generating hypervariability at the reactive center of serine proteases and their inhibitors are gene conversion followed by natural selection and natural selection for point mutation. One way to clarify the effects of these two mechanisms is to calculate separately the number of nonsynonymous substitutions and that of synonymous substitutions at the variable regions and at the conserved regions. Our data analysis shows that not only the number of nonsynonymous substitutions but also the number of synonymous substitutions at the variable regions exceed the corresponding numbers at the conserved regions. Thus gene conversion has provided needed variability at the variable regions of serine proteases and their inhibitors. Natural selection has helped perpetuate such variability.
The sequences of a conserved region in the A subunit of DNA gyrase corresponding to the quinolone resistance-determining region were determined for nine mycobacterial species and were compared. Although the nucleotide sequences were highly conserved, they clearly differentiated one species from another. The results of the phylogenetic analysis based on the sequences of the quinolone resistance-determining regions were compared with those provided by the 16S rRNA sequences. Deduced amino acid sequences were identical within the nine species except for amino acid 83, which was frequently involved in acquired resistance to quinolones in many genera, including mycobacteria. The presence at position 83 of an alanine for seven mycobacterial species (M. tuberculosis, M. bovis BCG, M. leprae, M. avium, M. kansasii, M. chelonae, and M. smegmatis) and of a serine for the two remaining mycobacterial species (M. fortuitum and M. aurum) correlated well with the MICs of ofloxacin for both groups of species, suggesting the role of this residue in intrinsic susceptibility to quinolones in mycobacteria.