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PubMed1993

Identification of human chromosome 9 specific genes using exon amplification.

We have recently developed a method, exon amplification, that is designed for isolation of exon sequences from genomic DNA. To assess the efficacy of this method we have analyzed cosmid genomic clones derived from human chromosome 9, and have cloned several products from this analysis. Approximately 63% of cosmids produced at least one product derived from functioning splice sites within the target genomic fragment, and in many cases multiple products were isolated. In addition, an easily identifiable class of false positives was produced from 56% of cosmids analyzed; these are readily eliminated from subsequent study. Sequence analysis and database searches revealed that the majority (87%) of the putative exon clones were unique, the remainder being derived from repetitive sequences. Analysis of sequence conservation by Southern blotting in addition to cDNA screening experiments suggested that most, if not all, of these unique sequences represent true exons. The results of these studies indicate that exon amplification is a rapid and reliable approach for isolation of exon sequences from mammalian genomic DNA.

Animals↗
PubMed2000

Neocentromeres and alpha satellite: a proposed structural code for functional human centromere DNA.

A comparative analysis of the conserved region of primate alpha satellite DNA and of a human neocentromere is reported. The two types of centromere DNA share structural features, possibly defining mitotically functional human centromere DNA. These features consist of double dyad symmetries of a particular size, as well as short conserved base motifs adjacent to the dyad symmetries. The possible importance of these features is supported by their presence also in the centromeres of yeast. The overall resemblance between primate alpha satellite DNA and yeast centromere DNA is further strengthened by the presence of a second shared structure. This structure consists of a single dyad symmetry incorporating a particular short base sequence. In the mitotically stable human neocentromere, this second symmetry is not found in connection with the double symmetry. However, the yeast homologue of the second symmetry is responsible for stability in meiosis I, rather than for mitotic stability. If the human homolog serves a similar function, it would be expendable, except for reproduction.

Animals↗
PubMed2005

Life table (survival) analysis to generate cumulative pregnancy rates in assisted reproduction: are we overestimating our success rates?

The variability in the numbers of treatment cycles couples may undertake with assisted reproductive technology (ART) and the length of time they may have to wait between successive cycles of treatment make the evaluation of treatment efficacy and prognosis complicated. The cumulative pregnancy rate using the life table method of analysis is being used more frequently to estimate the effectiveness of treatment. Although this approach is valid in some areas of infertility research, its use in ART is not appropriate, because the factors necessary for the analysis (particularly the scale for measuring the passage of time and lack of informative censoring) are not satisfied. Consequently, an overestimation of the effect of treatment is produced that may lead to biased decision making. Although there is no easy solution to this problem, several options for summarizing the outcome data are offered: pregnancy rate per cycle, time-limited analysis using proportions, conservative cycle-based cumulative pregnancy rate and real-time-based cumulative pregnancy rate. In this manner, more realistic information can be generated to counsel patients, evaluate the efficacy of treatments, compare rates among centres and guide the formulation of policies for infertility management and resource allocation.

Female↗
PubMed1986

Loss of the oncogene from human H-ras-1-transfected NIH/3T3 cells grown in the presence of excess methionine.

Mouse NIH/3T3 cells, transfected with T24 genomic DNA (J10 cells), carry the human H-ras-1 (hu-H-ras-1) oncogene stably integrated in the chromosomal DNA and display the transformed phenotype with a "criss-cross" morphology and capacity for anchorage-independent growth. When these cells were cultured in the presence of 25 mM DL-methionine, they lost their transformed phenotype; the characters of the normal phenotype (before transfection) reappeared, even though cell viability, as measured by the cloning potential on a solid support, was fully conserved. Southern blot analysis showed that cells continuously propagated in methionine-supplemented medium (J10met), unlike cells grown in normal medium, progressively segregated the hu-H-ras-1 gene. The progressively fewer cells still retaining the oncogene could be selected by inoculation of J10met cells into nude (nu/nu) mice of Swiss background. After prolonged methionine treatment, however, no such oncogene-positive cells could be detected, even when the excess methionine was withdrawn from the culture medium after 105 days. Conclusive evidence for the loss of the hu-H-ras-1 oncogene sequence was provided by subcloning the J10met cells. The oncogene sequence could no longer be found, and a fraction of subclones was no longer tumorigenic when assayed in nude mice. Other subclones generated late tumors negative for the hu-H-ras-1 transforming sequence, and a small fraction of subclones gave rise to rapidly growing early tumors that were also negative for the transforming sequence. The question of how methionine can induce the phenotypic and genetic change from malignancy to normality is discussed.

Animals↗
PubMed1993

Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development.

To begin to examine the function of genes that control early development in the hindbrain, we have screened an embryonic zebrafish cDNA library with a murine krox-20 gene probe that contained the conserved zinc finger regions. We have isolated two overlapping cDNAs, zf187 and zf201 which are homologues of the murine krox-20 gene. The N-terminal of the longest cDNA (zf201) contains two acidic regions identical to those of the murine krox-20. This indicates that the functional organisation of these proteins is probably conserved. Northern Blot analysis identified a single transcript of 2.0 kb. Wholemount in situ hybridisation established that expression of the zebrafish gene (krx-20) first appears at 100% epiboly as a single anterior domain of the prospective neuroepithelium, followed very soon after by a second more posterior domain. The alternating pattern of expression of this gene in rhombomeres(r) r3 and r5 is apparent by 12 hr post-fertilisation, that is prior to the morphological appearance of the rhombomeres. Around 14 hr neural crest migration begins from the dorsal surface of r5, moving caudally into r6 and then ventrally towards the pharyngeal arches. Crest migration is not apparent at or after 16 hr. No neural crest migration was observed from r3. Expression of krx-20 is down regulated firstly in r3 around 26 hr and later in r5 around 30 hr.

Amino Acid Sequence↗
PubMed2000

Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum.

We report the production, purification and characterization of a DNA ligase encoded by the thermophilic archaeon Methanobacterium thermoautotrophicum. The 561 amino acid MTH: ligase catalyzed strand-joining on a singly nicked DNA in the presence of a divalent cation (magnesium, manganese or cobalt) and ATP (K(m) 1.1 microM). dATP can substitute for ATP, but CTP, GTP, UTP and NAD(+) cannot. MTH: ligase activity is thermophilic in vitro, with optimal nick-joining at 60 degrees C. Mutational analysis of the conserved active site motif I (KxDG) illuminated essential roles for Lys251 and Asp253 at different steps of the ligation reaction. Mutant K251A is unable to form the covalent ligase-adenylate intermediate (step 1) and hence cannot seal a 3'-OH/5'-PO(4) nick. Yet, K251A catalyzes phosphodiester bond formation at a pre-adenylated nick (step 3). Mutant D253A is active in ligase-adenylate formation, but defective in activating the nick via formation of the DNA-adenylate intermediate (step 2). D253A is also impaired in phosphodiester bond formation at a pre-adenylated nick. A profound step 3 arrest, with accumulation of high levels of DNA-adenylate, could be elicited for the wild-type MTH: ligase by inclusion of calcium as the divalent cation cofactor. MTH: ligase sediments as a monomer in a glycerol gradient. Structure probing by limited proteolysis suggested that MTH: ligase is a tightly folded protein punctuated by a surface-accessible loop between nucleotidyl transferase motifs III and IIIa.

Adenosine Triphosphate↗
PubMed2001

On the molecular discrimination between adenine and guanine by proteins.

The molecular recognition and discrimination of adenine and guanine ligand moieties in complexes with proteins have been studied using empirical observations on carefully selected crystal structures. The distribution of protein folds that bind these purines has been found to differ significantly from that across the whole PDB, but the most populated architectures and folds are also the most common in three genomes from the three different domains of life. The protein environments around the two nucleic acid bases were significantly different, in terms of the propensities of amino acid residues to be in the binding site, as well as their propensities to form hydrogen bonds to the bases. Plots of the distribution of protein atoms around the two purines clearly show different clustering of hydrogen bond donors and acceptors opposite complimentary acceptors and donors in the rings, with hydrophobic areas below and above the rings. However, the clustering pattern is fuzzy, reflecting the variety of ways that proteins have evolved to recognise the same molecular moiety. Furthermore, an analysis of the conservation of residues in the protein chains binding guanine shows that residues in contact with the base are in general better conserved than the rest of the chain.

Adenine↗
PubMed1997

Selection on the protein-coding genes of the TBE1 family of transposable elements in the ciliates Oxytricha fallax and O. trifallax.

TBE1s are "cut-and-paste" transposable elements found in high copy number in the germline genomes of the ciliates Oxytricha fallax and O. trifallax. TBE1 "family" sequence (sequence of mixed polymerase chain reaction products generated using primers that match roughly half the TBE1s in host whole-cell DNA) was obtained from both host species. Although family sequence autoradiograms represent thousands of different elements, they are as legible as those representing corresponding sequences of a single TBE1, implying that ideal polymorphisms are rare within the genes examined. Nucleotide polymorphisms among TBE1s (indicated by ambiguities in family sequence) are far more common at third than at first or second positions of codons of genes, implying that selection has conserved the amino acid sequences of these genes in the majority of TBE1s. Portions of the transposase gene and another TBE1 gene have been sequenced from 10 individual TBE1s. None of these portions is interrupted by stop codons or frameshifts, and, for both genes, pairwise comparisons of these sequences show that nonsynonymous differences are significantly less common than synonymous differences, again implicating conservative selection Phylogenetic analysis shows that multiple divergent lineages of TBE1s have evolved under this selection within O. fallax. All these results are unexpected for cut-and-paste transposons in eukaryotic hosts: since transposase encoded by intact elements presumably acts in trans, it can duplicate mutant copies (those that do not encode functional transposase) found in the same genome, and thus no selection is expected to maintain the transposase gene. The selection demonstrated here could act at transposition (if functional TBE1s are preferentially transposed) or at the level of the host (if the host's fitness depends on functional TBE1 genes). TBE1-encoded proteins might be responsible for the precise excision of TBE1s that occurs during development of the host somatic nucleus; selection on hosts for uninterrupted somatic genes would then translate into selection for TBE1 protein-coding competence. We suggest a method for distinguishing between these two classes of explanations by finding and analyzing divergent alleles of ancestral transposable element insertions.

Animals↗
PubMed1999

Selection at the wobble position of codons read by the same tRNA in Saccharomyces cerevisiae.

The transfer RNA gene complement of Saccharomyces cerevisiae was utilized for a whole-genome analysis of the deviation from a neutral usage of pyrimidine-ending cognate codons, that is, codons read by a single tRNA species having either inosine or guanosine as the first anticodon base. Mutational pressure at the wobble position was estimated from the base composition of the noncoding portion of the yeast genome. The selective pressure for translational efficiency was inferred from the degree of codon adaptation to tRNA gene redundancy and from mRNA abundance data derived from yeast transcriptome analysis. Amino acid conservation in orthologous comparisons with wholly sequenced microbial genomes was used to estimate translational accuracy requirements. A close correspondence was observed between the usage of wobble position pyrimidines and the frequency predicted by mutational bias. However, in the case of four cognate pairs (Gly: ggu/ggc; Asn: aau/aac; Phe: uuu/uuc; Tyr: uau/ uac) all read by guanosine-starting anticodons, we found evidence for a strong selective pressure driven by translational efficiency. Only for the glycine pair, wobble pyrimidine choice also appears to fulfill a translational accuracy requirement. Wobble pyrimidine selection is strictly related to the number of hydrogen bonds formed by alternative cognate codons: whenever a different number of hydrogen bonds can be formed at the wobble position, there is selection against six- or nine-hydrogen-bonded codon-anticodon pairs. Our results indicate that an intrinsic codon preference, critically dependent on the stability of codon-anticodon interaction and mainly reflecting selection for the optimization of translational efficiency, is built into the translational apparatus.

Codon↗
PubMed1994

Molecular evolution of a multigene family in group A streptococci.

The emm genes are members of a gene family in group A streptococci (GAS) that encode for antiphagocytic cell-surface proteins and/or immunoglobulin-binding proteins. Previously sequenced genes in this family have been named "emm," "fcrA," "enn," "arp," "protH," and "mrp"; herein they will be referred to as the "emm gene family." The genes in the emm family are located in a cluster occupying 3-6 kb between the genes mry and scpA on the chromosome of Streptococcus pyogenes. Most GAS strains contain one to three tandemly arranged copies of emm-family genes in the cluster, but the alleles within the cluster vary among different strains. Phylogenetic analysis of the conserved sequences at the 3' end of these genes differentiates all known members of this family into four evolutionarily distinct emm subfamilies. As a starting point to analyze how the different subfamilies are related evolutionarily, the structure of the emm chromosomal region was mapped in a number of diverse GAS strains by using subfamily-specific primers in the polymerase chain reaction. Nine distinct chromosomal patterns of the genes in the emm gene cluster were found. These nine chromosomal patterns support a model for the evolution of the emm gene family in which gene duplication followed by sequence divergence resulted in the generation of four major-gene subfamilies in this locus.

Amino Acid Sequence↗
PubMed2026

Potato purple top phytoplasma infection induces autophagy-associated lipid dynamics that support pathogen proliferation.

Phytoplasmas are unculturable, phloem-restricted bacterial pathogens responsible for devastating diseases in crops and ornamentals worldwide. Their mechanism for nutrient acquisition from host plants remains largely unknown. This study demonstrated that infection with potato purple top phytoplasma induced extensive remodeling of lipid metabolism in tomato plants, closely linked to autophagy activation. Western blot and confocal analyses revealed increased ATG8 lipidation and autophagosome formation at endoplasmic reticulum stress sites, alongside the redistribution of lipid droplets toward phytoplasma cells. Lipidomic profiling showed a decline in chloroplast galactolipids and phospholipids with a concomitant rise in triacylglycerol, indicating accelerated membrane turnover and neutral lipid sequestration. Transmission electron microscopy further revealed frequent spatial proximity between lipid droplets and phytoplasmas. Inhibition of autophagy with 3-methyladenine blocked lipid droplet breakdown, disrupted endoplasmic reticulum organization, and reduced phytoplasma titers, suggesting that host autophagy contributes to phytoplasma proliferation. In addition, genome analysis identified a conserved phytoplasma-encoded alpha/beta hydrolase (potato purple top-lipase), predicted to be related to monoacylglycerol lipases. In vivo assays in yeast and Nicotiana benthamiana confirmed that potato purple top-lipase reduced neutral lipids, mainly triacylglycerol, and that catalytic triad mutations abolished activity. Because potato purple top-lipase lacks a predicted secretory signal peptide, it likely functions intracellularly within phytoplasma cells and may participate in the metabolism of lipid intermediates. These findings support a model in which phytoplasma infection is associated with host autophagy-associated lipid droplet mobilization and a phytoplasma lipase that may contribute to host-derived lipid resources, providing insight into potential nutrient acquisition strategies of phloem-restricted pathogens.

Autophagy↗
PubMed1999

Susceptibility of beta-lactamase to core amino acid substitutions.

To determine which amino acids in TEM-1 beta-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme. A comprehensive screening of these libraries identified several TEM-1 beta-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity. An examination of positions 66 and 76 in the class A beta-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other beta-lactamases. It is possible that position 76 varies in the gene family because beta-lactamase mutants with non-conservative substitutions at position 76 retain partial function. In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function. By determining the beta-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions. An analysis of the Protein Data Bank files for three class A beta-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position.

Amino Acid Sequence↗
PubMed2005

Modelling of activation processes for GR-280 graphite at Ignalina NPP.

Ignalina Nuclear Power Plant (INPP) operates two RBMK-1500 water-cooled graphite-moderated channel-type power reactors. The total mass of graphite in the cores of both units at INPP is about 3600 tons. Modelling of activation processes in the reactor's structural materials is necessary for decommissioning planning, because large amounts of activated structural materials (graphite, stainless steel, concrete, etc.) should be managed as radioactive waste. Knowledge of radiological characteristics and a radioactive inventory of irradiated materials are essential in planning of the decommissioning processes. The purpose of this work was to perform conservative neutron activation analysis for decommissioning purposes of INPP. ORIGEN computer code was used for the calculations. Activity levels were calculated for different nuclides present in the graphite and estimates were made how these activity levels depend on irradiation time, neutron flux value and other parameters. Obtained results were compared with the data available from other investigations for GR-280 graphite.

Computer Simulation↗
PubMed1998

The omega sequence of VirD2 is important but not essential for efficient transfer of T-DNA by Agrobacterium tumefaciens.

The VirD2 protein of Agrobacterium tumefaciens contains defined sequences necessary for processing and transferring the T-DNA during transformation of plant cells. We performed a mutational analysis of the conserved omega sequence of VirD2, whose role has proven to be difficult to elucidate so far. In this report, we show that a deletion of these 5 amino acids or their replacement by 5 glycines reduced T-DNA transfer considerably, compared with wild type, demonstrating that the omega sequence is important for the efficient transfer of T-DNAs. However, the efficiency and pattern of integration of the T-DNAs were not affected by any modifications of the omega sequence. The importance of the C terminus of VirD2 for T-DNA transfer is discussed.

Agrobacterium tumefaciens↗
PubMed2001

Diversification of neurotoxins by C-tail 'wiggling': a scorpion recipe for survival.

The structure of bioactive surfaces of proteins is a subject of intensive research, yet the mechanisms by which such surfaces have evolved are largely unknown. Polypeptide toxins produced by venomous animals such as sea anemones, cone snails, scorpions, and snakes show multiple routes for active site diversification, each maintaining a typical conserved scaffold. Comparative analysis of an array of genetically related scorpion polypeptide toxins that modulate sodium channels in neuronal membranes suggests a unique route of toxic site diversification. This premise is based on recent identification of bioactive surfaces of toxin representative of three distinct pharmacological groups and a comparison of their 3-dimensional structures. Despite their similar scaffold, the bioactive surfaces of the various toxins vary considerably, but always coincide with the molecular exterior onto which the C-tail is anchored. Superposition of the toxin structures indicates that the C-tails diverge from a common structural start point, which suggests that the pharmacological versatility displayed by these toxins might have been achieved along evolution via structural reconfiguration of the C-tail, leading to reshaping of new bioactive surfaces.

Amino Acid Sequence↗
PubMedDate not supplied

Intestinal obstruction in Crohn's disease in childhood.

Out of 540 children and adolescents from the Multicenter Pediatric Crohn's Disease Study Group, 42 patients presented with decompensated intestinal obstruction. In 26 patients only one intestinal obstruction occurred, and in 16 children up to five intestinal obstructions occurred. Conservative measures were successful in 37 of 72 episodes of intestinal obstruction (51.4%), while 19 of 42 patients underwent emergency surgery (45.2%) and 16 of 42 (38.1%) were operated on following conservative treatment. From analysis of clinical, intraoperative, and histological findings in surgically treated patients, criteria for emergency surgery and elective surgery of intestinal obstruction in Crohn's disease are derived. One patient with adenocarcinoma of the large bowel, presenting with recurrent obstruction, signals caution in delaying proper diagnosis.

Adolescent↗
PubMed2002

Traumatic intracerebellar hemorrhage: clinicoradiological analysis of 81 patients.

OBJECTIVE: We report 81 patients with a traumatic intracerebellar hemorrhagic contusion or hematoma managed between 1996 and 1998 at 13 Italian neurosurgical centers. METHODS: Each center provided data about patients' clinicoradiological findings, management, and outcomes, which were retrospectively reviewed. RESULTS: A poor result occurred in 36 patients (44.4%). Forty-five patients (55.6%) had favorable results. For the purpose of data analysis, patients were divided into two groups according to their admission Glasgow Coma Scale (GCS) scores. In Group 1 (39/81 cases; GCS score, > or =8), the outcome was favorable in 95% of cases. In Group 2 (42/81 cases; GCS score, <8), the outcome was poor in 81% of cases. Twenty-seven patients underwent posterior fossa surgery. Factors correlating with outcome were GCS score, status of the basal cisterns and the fourth ventricle, associated supratentorial traumatic lesions, mechanism of injury, and intracerebellar clot size. Multivariate analysis showed significant independent prognostic effect only for GCS score (P = 0.000) and the concomitant presence of supratentorial lesions (P = 0.0035). CONCLUSION: This study describes clinicoradiological findings and prognostic factors regarding traumatic cerebellar injury. A general consensus emerged from this analysis that a conservative approach can be considered a viable, safe treatment option for noncomatose patients with intracerebellar clots measuring less than or equal to 3 cm, except when associated with other extradural or subdural posterior fossa focal lesions. Also, a general consensus was reached that surgery should be recommended for all patients with clots larger than 3 cm. The pathogenesis, biomechanics, and optimal management criteria of these rare lesions are still unclear, and larger observational studies are necessary.

Adolescent↗
PubMed1991

The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses.

Representative amino acid sequences of the RNA-dependent RNA polymerases of all groups of positive-strand RNA viruses were aligned hierarchiacally, starting with the most closely related ones. This resulted in delineation of three large supergroups. Within each of the supergroups, the sequences of segments of approximately 300 amino acid residues originating from the central and/or C-terminal portions of the polymerases could be aligned with statistically significant scores. Specific consensus patterns of conserved amino acid residues were derived for each of the supergroups. The composition of the polymerase supergroups was as follows. I. Picorna-, noda-, como-, nepo-, poty-, bymo-, sobemoviruses, and a subset of luteoviruses (beet western yellows virus and potato leafroll virus). II. Carmo-, tombus-, dianthoviruses, another subset of luteoviruses (barley yellow dwarf virus), pestiviruses, hepatitis C virus (HCV), flaviviruses and, unexpectedly, single-stranded RNA bacteriophages. III. Tobamo-, tobra-, hordei-, tricornaviruses, beet yellows virus, alpha-, rubi-, furoviruses, hepatitis E virus (HEV), potex-, carla-, tymoviruses, and apple chlorotic leaf spot virus. An unusual organization was shown for corona- and torovirus polymerases whose N-terminal regions were found to be related to the respective domains of supergroup I, and the C-terminal regions to those of the supergroup III polymerases. The alignments of the three polymerase supergroups were superimposed to produce a comprehensive final alignment encompassing eight distinct conserved motifs. Phylogenetic analysis using three independent methods of three construction confirmed the separation of the positive-strand RNA viral polymerases into three supergroups and revealed some unexpected clusters within the supergroups. These included the grouping of HCV and the pestiviruses with carmoviruses and related plant viruses in super-group II, and the grouping of HEV and rubiviruses with furoviruses in supergroup III.

Amino Acid Sequence↗