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At least 19 recordsLinked to original sources
PubMed2026

Nucleotide Combination Proportions Across Algae, Monocotyledons and Dicotyledons: Insights into Plant Genome Evolution.

Plant evolution started with unicellular algae, gradually evolving multicellularity and terrestrial colonization. These evolutionary events were accompanied by the interplay of chromosome polyploidization, rearrangement, gene loss, and point mutation. We counted the proportion of nucleotide combinations in the genome sequences of 64 sequenced plants, and analyzed the significant difference in these nucleotide combination proportions among algae, monocotyledons and dicotyledons. The correlation of highly significant different and no significant different nucleotide combinations was analyzed respectively. Nucleotide combinations and their reverse complementary sequence proportions were analyzed in different functional regions of the genome. These results reveal that some nucleotide combinations are subject to strict selection, and these combinations have a higher proportion in the CDS regions and lower proportion in the intergenic regions. Meanwhile, there are some nucleotide combinations that are under less selective pressure, and these combinations have a higher proportion in the intergenic regions and lower proportion in the CDS regions. Cluster analysis based on trinucleotide to octanucleotide combination proportions reveals that plant genome evolution is accompanied by clade-wide differentiation of genome-wide nucleotide composition patterns, in addition to well-documented chromosomal polyploidization, structural rearrangement and gene loss events. We analyzed the changes in the proportion of nucleotide combinations at the genome level in 64 sequenced plants, providing a new idea for studying genome evolution in the plant kingdom.

comparative genomics↗
PubMed2002

Analysis, quantification, and evolutionary consequences of HIV-1 in vitro recombination.

HIV-1 recombination was studied in vitro by viral cocultivation of four combinations of strains of subtypes B, D, and F. Viral cocultivations were performed in MT-4 cells and maintained for 22 days. The parental and recombinant forms were quantified by a specific PCR system in an env fragment of 2500 nucleotides. On day 5, there was a close correlation between the proportion of recombination and the genetic distance between strains. In three of the four viral combinations studied, a steady increase in the proportion of recombinant genomes was observed over time. This rise coincided with the progressive loss of one of the parental strains, resulting in less diverse viral populations. Nucleotide sequencing of biological recombinant clones from the B/D cocultivation revealed a higher number of recombination events in pol than in env gene, and an increasing number of crossovers per clone with time.

Coculture Techniques↗
PubMed1993

Antibody engineering by parsimonious mutagenesis.

The human monoclonal antibody (humAb) problem has largely been solved with the aid of the polymerase chain reaction (PCR) [Larrick et al., Bio/Technology 7 (1989a) 934-938; Larrick et al., Biochem. Biophys. Res. Commun. 160 (1989b) 1250-1256; Chiang et al., BioTechniques 7 (1989) 360-366]. Phage display has now made it possible to recover humAb with primary response level affinities (approx. 10(6) M-1) for virtually any antigen (including self antigens) from comprehensive libraries of B-cell repertoires from non-immunized humans [Marks et al., J. Mol. Biol. 222 (1991) 581-597; Marks et al., Bio/Technology 10 (1992) 779-783; Griffiths et al., EMBO J. 12 (1993) 725-734]. This means that the goal of therapeutic humAb without immunization is within reach. However, in order to achieve the affinities generally required for therapeutic use (> or = 10(9) M-1), reliable methods will be needed to complete the affinity maturation process in vitro. Available X-ray crystallographic data and energy calculations indicate that only a fraction of the substantial contact surface between the Ab and protein antigens contribute significantly to affinity. Thus, the remaining contact surface presents multiple opportunities to develop additional high-affinity contacts, needing only a means to identify them. To this end, we have developed a computer-assisted method for oligodeoxyribonucleotide-directed scanning mutagenesis, called parsimonious mutagenesis (PM), whereby all three complementarity-determining regions (CDR) of a variable region (V-region) gene can be simultaneously and thoroughly searched for improved variants in libraries of manageable size. These libraries are made with low-redundancy 'doping' codons and biased nucleotide (nt) mixtures designed to maximize the abundance of combining sites with predetermined proportions of preselected sets of alternative amino acids (aa). This allows the library to 'probe' the surface of the antigen one or a few aa residues at a time with a wide selection of aa side chains to search out and identify new high-affinity contacts. In addition to affinity maturation in vitro, PM can also be used to remove unwanted cross-reactivities and to 'reshape' rodent mAb for human therapeutic use.

Amino Acid Sequence↗
PubMed1976

Dissociation and reassociation of the phosphorylated and nonphosphorylated forms of adenosine 3':5' -monophosphate-dependent protein kinase from bovine cardiac muscle.

Adenosine 3':5' -monophosphate (cyclic AMP) -dependent protein kinase from bovine heart muscle catalyzes the phosphorylation of its regulatory, cyclic AMP-binding subunit. Phosphorylation enhances net dissociation of the enzyme by cyclic AMP. Chromatography on omega-aminohexyl-agarose was used to study the effects of phosphorylation on cyclic AMP binding and subunit dissociation and reassociation. This method permitted rapid separation of the catalytic subunit from the cyclic AMP -binding protein and holoenzyme. Phospho- and dephosphoprotein kinases were found to dissociate to the same extent at any given concentration of cyclic AMP and completely at saturation. At equilibrium, the amount of cyclic AMP bound was the same for both forms of enzyme and was directly proportional to the degree of dissociation of the holoenzyme. In the absence of cyclic AMP, phospho- and dephospho-cyclic AMP-binding proteins reassociated completely with the catalytic subunit. However, the rate of reassociation of the dephospho-cyclic AMP-binding protein was at least 5 times greater than the phospho-cyclic AMP-binding protein. Retardation of reassociation was directly proportional to the extent of phosphorylation. We conclude that the degree to which the cyclic AMP-binding protein is phosphorylated markedly affects its intrinsic ability to combine with the catalytic subunit to regenerate the inactive cyclic nucleotide-dependent kinase and that the state of phosphorylation of this subunit may be important in detemining the proportion of dissociated (active) and reassociated (inactive) protein kinase at any given time.

Animals↗
PubMed2003

Tightly linked di- and tri-nucleotide microsatellites do not evolve in complete independence: evidence from linked (TA)n and (TAA)n microsatellites of chickpea (Cicer arietinum L.).

In order to understand the dynamics of microsatellite evolution, we have studied allelic variation at a closely linked (TA)(n) and (TAA)(n) microsatellite loci in 114 land races of chickpea ( Cicer arietinum L.), sampled worldwide. These two loci are separated by 27 bp. The two loci showed a very high degree of polymorphism and hence the combined length with the genetic diversity of 0.93, 0.90 and 0.98 for (TAA)(n), (TA)(n) and the combined length, respectively. Using the variation data at the linked loci, a standardized index of linkage disequilibrium was also computed ( I(S)(A)=0.092), which tests the null hypothesis of no linkage and was significant, indicating the presence of linkage disequilibrium. Furthermore, the dynamics of allelic variation showed that there is a threshold combined length, below which both (TAA)(n) and (TA)(n) loci evolve independently, and above which, if one locus increase in size, the other closely linked locus has a tendency to decrease its size and vice versa, without change in the overall ratio of (TAA)(n) and (TA)(n) allele sizes at the region. This result indicates that there are processes in the cell, which 'read' the combined size of the two loci both for proportion and length and determine the direction of tightly linked di- and tri-nucleotide repeat evolution.

Alleles↗
PubMedDate not supplied

ADAPT: a molecular mechanics approach for studying the structural properties of long DNA sequences.

We describe an original approach to determining sequence-structure relationships for DNA. This approach, termed ADAPT, combines all-atom molecular mechanics with a multicopy algorithm to build nucleotides that contain all four standard bases in variable proportions. These nucleotides enable us to search very rapidly for base sequences that energetically favor chosen types of DNA deformation or chosen DNA-protein or DNA-ligand interactions. Sequences satisfying the chosen criteria can be found by energy minimization, combinatorial sequence searching, or genome scanning, in a manner similar to the threading approaches developed for protein structure prediction. In the latter case, we are able to analyze roughly 2000 base pairs per second. Applications of the method to DNA allomorphic transitions, DNA deformation, and specific DNA interactions are presented.

Algorithms↗
PubMed1983

Urinary cyclic AMP and cyclic GMP in rats given three different concentrations of cadmium in the diet for one year.

Male and female weanling rats of the SPF Fisher strain were fed purified diets containing three different levels of cadmium (0.008, 0.3, 30 micrograms Cd/g feed). Urine was collected from each animal 1 year after the start of the experiment, and cyclic AMP and cyclic AMP in the urine were measured by radioimmunoassay. Urinary excretion of cyclic AMP and GMP changed in proportion to the intake of Cd. The ratio of cyclic AMP: cyclic GMP, which combined the changes of these two cyclic nucleotides, increased with Cd intake, and these relationships were statistically significantly different in males and females.

Animals↗
PubMed1982

The double-stranded RNA genome segments of cytoplasmic polyhedrosis virus are independently transcribed.

The in vitro transcription of the genome segments of insect cytoplasmic polyhedrosis virus was examined for a possible mechanism of coordinate control. Initiation of the first transcription cycle, primed by preincubation in the absence of CTP, was synchronous for all 10 viral genome segments. The time required for the completion of each genome transcript was proportional to the chain length, and all 10 mRNA's were synthesized in molar amounts inversely proportional to their molecular weights. The rate of chain elongation for all mRNA's was approximately 6 nucleotides per s. The combined results indicate that each of the viral genome segments is transcribed independently and suggest that RNA chain elongation, not initiation, is rate limiting for cytoplasmic polyhedrosis virus mRNA synthesis in vitro.

Animals↗
PubMed1999

Covalent crosslinks introduced via a triple helix-forming oligonucleotide coupled to psoralen are inefficiently repaired.

Triple helix-forming oligonucleotides (TFOs) represent potentially powerful tools to artificially modulate gene activity. In particular, they can be used to specifically introduce a lesion into a selected target sequence: interstrand crosslinks and monoadducts can be introduced via TFOs coupled to psoralen. The efficiency of these strategies depends on the cell ability to repair these lesions, an issue which is still controversial. Here we show, using psoralen-coupled TFOs and the yeast as a convenient cellular test system, that interstrand crosslinks are quantitatively poorly repaired, resulting in an efficient modification of target gene activity. In addition, these lesions result in the introduction of mutations in a high proportion of cells. We show that these mutations are generated by the Error-Prone Repair pathway, alone or in combination with Nucleotide Excision Repair. Taken together, these results suggest that TFOs coupled to psoralen could be used to inactivate a gene with significant efficiency.

Adenosine Triphosphatases↗
PubMed2025

Interpreting the CTCF-mediated sequence grammar of genome folding with AkitaV2.

Interphase mammalian genomes are folded in 3D with complex locus-specific patterns that impact gene regulation. CTCF (CCCTC-binding factor) is a key architectural protein that binds specific DNA sites, halts cohesin-mediated loop extrusion, and enables long-range chromatin interactions. There are hundreds of thousands of annotated CTCF-binding sites in mammalian genomes; disruptions of some result in distinct phenotypes, while others have no visible effect. Despite their importance, the determinants of which CTCF sites are necessary for genome folding and gene regulation remain unclear. Here, we update and utilize Akita, a convolutional neural network model, to extract the sequence preferences and grammar of CTCF contributing to genome folding. Our analyses of individual CTCF sites reveal four predictions: (i) only a small fraction of genomic sites are impactful; (ii) impact is highly dependent on sequences flanking the core CTCF binding motif; (iii) core and flanking nucleotides contribute largely additively to the overall impact of a site; (iv) sites created as combinations of different core and flanking sequences have impacts proportional to the product of their average impacts, i.e. they are broadly compatible. Our analysis of collections of CTCF sites make two predictions for multi-motif grammar: (i) insulation strength depends on the number of CTCF sites within a cluster, and (ii) pattern formation is governed by the orientation and spacing of these sites, rather than any inherent specialization of the CTCF motifs themselves. In sum, we present a framework for using neural network models to probe the sequences instructing genome folding and provide a number of predictions to guide future experimental inquiries.

CCCTC-Binding Factor↗
PubMed1983

Modulation of deoxycytidine metabolism in vivo with high-dose thymidine in mice.

The question of whether selective biochemical modulation of deoxycytidine (dCyd) metabolism by thymidine (dThd) could be quantitated in vivo was addressed. Plasma clearance of [5-3H]dCyd was altered significantly by prior administration (2 hr) of dThd (3,600 mg/kg). The plasma half-life (beta-phase) of [5-3H]dCyd was raised approximately fourfold by dThd. Although dThd consistently caused an increase in the [5-3H]dCyd incorporated into the DNA of the spleen, duodenum, and femur marrow, there was no clear-cut difference between normal (BALB/c X DBA/2)F1 mice and L1210 tumor-bearing (BALB/c X DBA/2)F1 mice that could be translated into a selective advantage. Analysis of tritiated nucleotides in the spleens of normal and L1210 tumor-bearing mice indicated a greater incorporation of [5-3H]dCyd into the dCyd nucleotide pools of L1210 tumor-bearing mice than of normal mice. An analysis with the use of the proportionality constant kappa representing the combined effects of transport, pool sizes, and metabolic incorporation of [5-3H]dCyd into DNA showed that kappa decreased in each organ studied after dThd administration.

Animals↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed1986

Misincorporation by AMV reverse transcriptase shows strong dependence on the combination of template and substrate nucleotides.

We have carried out a systematic investigation of the efficiency of misincorporation by Avian Myeloblastosis Virus reverse transcriptase with all possible combinations of dNTP substrate, template nucleotide, and the nucleotide at the 3' terminus of the primer. A series of synthetic oligonucleotide primers were annealed to single stranded M13 DNA templates, and a single dNTP was misincorporated at the primer 3' end using AMV reverse transcriptase. The proportion and pattern of misincorporation and incorporation in all 64 situations was assayed using [5'-32p] labelled primers, and the products were separated on denaturing polyacrylamide gels. Correct incorporations occurred more readily than misincorporations. The efficiency of misincorporation depended on the individual primer, but, comparing primers, a clear dependence on the template nucleotide was observed for the preferential misincorporation of different dNTPs. The exact combination of template and dNTP was important; although purine:pyrimidine (dNTP substrate:template nucleotide) and pyrimidine:purine misincorporations occurred comparatively readily, some pyrimidine:pyrimidine and purine:purine reactions were equally efficient and yet others were never seen to occur. Some misincorporations were facilitated by subsequent correct incorporations, but despite this our results suggest that the level of misincorporation is limited by the rate of reaction and enzyme inactivation rather than by exonuclease activity.

Avian Myeloblastosis Virus↗
PubMed1991

Detection of complex alleles by direct analysis of DNA heteroduplexes.

DNA molecules derived from three alleles of the HLA-DRB3 locus and differing from each other at several nucleotide sites were denatured and cross-hybridized. Each allelic combination was found to generate a pair of heteroduplexes of different mobility. Their retardation as compared to homoduplexes was proportional to the number of mismatches. In each heteroduplexes pair the component possessing the highest number of Pyr-Pyr oppositions was the most retarded. The results are those predicted by a theoretical model implying a correlation between base-pair opening and bending of the DNA double helix. These observations introduce a new HLA typing method at the genomic level and indicate an experimental approach to the analysis of the superhelical DNA conformation as related to different types of base oppositions.

Alleles↗
PubMed2006

CrossLink: visualization and exploration of sequence relationships between (micro) RNAs.

CrossLink is a versatile tool for the exploration of relationships between RNA sequences. After a parametrization phase, CrossLink delegates the determination of sequence relationships to established tools (BLAST, Vmatch and RNAhybrid) and then constructs a network. Each node in this network represents a sequence and each link represents a match or a set of matches. Match attributes are reflected by graphical attributes of the links and corresponding alignments are displayed on a mouse-click. The distributions of match attributes such as E-value, match length and proportion of identical nucleotides are displayed as histograms. Sequence sets can be highlighted and visibility of designated matches can be suppressed by real-time adjustable thresholds for attribute combinations. Powerful network layout operations (such as spring-embedding algorithms) and navigation capabilities complete the exploration features of this tool. CrossLink can be especially useful in a microRNA context since Vmatch and RNAhybrid are suitable tools for determining the antisense and hybridization relationships, which are decisive for the interaction between microRNAs and their targets. CrossLink is available both online and as a standalone version at http://www-ab.informatik.uni-tuebingen.de/software.

Computer Graphics↗
PubMed1983

Simultaneous extraction and combined bioluminescent assay of NAD+ and NADH.

A new method for extracting pyridine nucleotides from tissue samples at room temperature that allows the simultaneous extraction of both the oxidized and reduced nucleotide when using a 70% buffered ethanol solution as the extractant has been developed. The extraction efficiencies for NAD+ and NADH were 91 and 102%, respectively. The extraction method was followed by a combined bioluminescent assay of both nucleotides. A bacterial bioluminescent system, which included luciferase and low levels of a NADH-specific oxidoreductase, was used to produce a constant light intensity directly proportional to the amount of NADH in the tissue extract sample. When the NADH had been measured, the NAD+ present in the extract was enzymatically converted to NADH by the addition of alcohol dehydrogenase, after which the second increase in light level was recorded. The sensitivity of the bioluminescent assay presented here is 5 X 10(-14) mol NADH or NAD+ per assay.

Alcohol Oxidoreductases↗
PubMed1996

Sequence simplicity and evolution of the 3' untranslated region of the histone H1o gene.

The H10 gene has a long 3' untranslated region (3'UTR) of 1,125 nucleotides in the rat and 1,310 in humans. Analysis of the sequences shows that they have features of simple DNA that suggest involvement of replication slippage in their evolution. These features include the length imbalance between the rat and human sequences; the abundance of single-base repeats, two-base runs and other simple motifs clustered along the sequence; and the presence of single-base repeat length polymorphisms in the rat and mouse sequences. Pairwise comparisons show numerous short insertions/deletions, often flanked by direct repeats. In addition, a proportion of short insertions/deletions results from length differences in conserved single-base repeats. Quantification of the sequence simplicity shows that simple sequences have been more actively incorporated in the human lineage than in the rodent lineage. The combination of insertions/deletions and nucleotide substitutions along the sequence gives rise to three main regions of homology: a highly variable central region flanked by more conserved regions nearest the coding region and the polyA addition site.

Animals↗
PubMed1990

Detection of deletions, insertions and single nucleotide substitutions in cloned beta-globin genes and new polymorphic nucleotide substitutions in beta-globin genes in a Japanese population using ribonuclease cleavage at mismatches in RNA:DNA duplexes.

The applicability of ribonuclease cleavage at mismatches in RNA:DNA duplexes (RNase cleavage method) for determining nucleotide variant rates has been examined in a Japanese population. DNA segments of various lengths obtained from 4 different regions of a normal and 3 thalassemic cloned human beta-globin genes were inserted into transcription vectors. Sense and antisense RNA probes uniformly labeled with 32P were prepared. When RNA probes of 771 nucleotides (nt) or less were hybridized with cloned DNAs and the resulting duplexes were treated with a mixture of RNases A and T1, the length of products agreed with theoretical values. Twelve possible mismatches were examined. Since both sense and antisense probes were used, uncleavable mismatches such as G:T and G:G which were made from one combination of RNA and DNA strands could be converted to the cleavable C:A and C:C mismatches, respectively, by using the opposite combination. Deletions and insertions of 1 (G), 4 (TTCT), 5 (ATTTT) and 10 (ATTTTATTTT) nt were easily detected. A polymorphic substitution of T to C at position 666 of the second intervening sequence (IVS2-666) of the beta-globin gene was detected using genomic DNAs from cell lines established from the peripheral B lymphocytes of 59 unrelated Japanese from Hiroshima or those amplified by polymerase chain reaction (PCR). The frequency of the gene with C at the IVS2-666 (allele C) was 0.48 and that of the gene with T (allele T) was 0.52. The associations of the 2 alleles were in agreement with Hardy-Weinberg proportions. No contradiction to Mendelian inheritance was observed in the results obtained from 11 family studies. Two new polymorphic substitutions of C to A and A to T were detected at nucleotide positions 1789 and 1945 from the capping site, respectively, using genomic DNAs amplified by PCR. The feasibility of the RNase cleavage method combined with PCR for large-scale screening of variation in chromosomal DNA is discussed.

Base Composition↗