Extreme Divergence Research Articles

Accelerated Evolution in Inhibitor Domains of Porcine Elafin Family Members

Through the analysis of the porcine gene encoding the elastase inhibitor elafin, we demonstrated that there are at least three closely related members of the elafin family, and their genes have arisen by accelerated evolution. A porcine genomic DNA library was screened with a previously cloned human elafin cDNA probe, and several positive clones were obtained that can be distinguished by a combination of restriction enzymes. Sequence analysis of these clones revealed the presence of three homologous members whose genes, all consisting of three exons and two introns, are almost identical except the exon 2 sequences encoding the inhibitor domain called "WAP motif"; the intron sequences are related to each other with sequence similarities of 93-98%, whereas the exon 2 sequences exhibited only 60-77% similarities among the three members. The extreme divergence in the exon 2 sequences compared to the highly conserved intron sequences may be generated by accelerated mutations confined in a short stretch of the genes following recent duplication events of a single ancestral gene. An RNase protection assay indicated that the messages of the elafin family members are abundantly expressed in the trachea and intestine, suggesting that the most likely selective forces for the accelerated evolution are extrinsic proteinases produced by invasive microorganisms.

Molecular characterization of hybrid Tbp2 proteins from Neisseria meningitidis.

Transferrin-binding protein 2 (Tbp2) from Neisseria is an outer membrane-associated extracellular lipoprotein that is involved in iron capture within the infected host. The analysis of tbp2 clones isolated from various bacterial strains revealed extensive divergences throughout the open reading frame (ORF), with predicted amino acid (aa) sequences displaying 47% to 83% identity. Such a variability is likely to have resulted from the selective pressure exerted by the host immune system, but raises questions regarding the existing constraints for conservation of protein function. Indeed, the neisserial Tbp2s include a large structured domain, extending throughout the N-terminal half of the protein (approximately 270-290 aa), which is extremely stable and whose conformational integrity is required for efficient binding to human transferrin (hTf). In this work, a functional study of Tbp2s encoded by hybrid genes constructed by reassorting highly divergent tbp2 sequences in the region of the ORF encoding this structured domain was performed. The data demonstrate that the determinant intramolecular interactions allowing formation of a stable Tbp2 structure able to interact efficiently with hTf or/and that the Tbp2 residues involved in the interaction with hTf are not well conserved. However, a number of rearrangements appeared to generate genes encoding proteins which have retained structural stability and hTf-binding capacity. This suggested that despite the extreme aa sequence divergence and the conformational constraints, horizontal genetic exchanges, which are known to occur in neisserial populations, may have contributed significantly to the generation of sequence variation within tbp2 ORFs. The analysis of two tbp2 clones characterized in this work supports this hypothesis.

Acquisition of apparent DNA slippage structures during extensive evolutionary divergence of pcaD and catD genes encoding identical catalytic activities in Acinetobacter calcoaceticus

The pca operon from the Gram − bacterium Acinetobacter calcoaceticus encodes all of the enzymes required for catabolism of protocatechuate to common intermediary metabolites. This report presents the 2754-nucleotide (nt) sequence of a HindIII restriction fragment containing pcaD, the 801-bp gene encoding β-ketoadipate enol-lactone hydrolase I. The deduced primary structure of A. calcoaceticus PcaD shares 44% amino acid (aa) sequence identity with the aligned primary structure of CatD (β-ketoadipate enol-lactone hydrolase II) from the same organism, and the overall nt sequence identity of the two genes is 51.8%. In the 56% of the genes where selection for identical aa residues was not imposed, pcaD and catD have diverged so extensively that nt sequence identity of the aligned segments is only 28.2%; the G + C contents of these segments from the respective genes differ by 8%. Conserved within the aligned PcaD and CatD aa sequences is a Ser residue corresponding to the nucleophile within the α/β-fold of many hydrolytic enzymes. In this region of primary structure, PcaD and CatD appear to have maintained some different aa sequences derived from a common ancestor. Conservation of the different aa sequences during extreme evolutionary divergence suggests that separate segments of primary structure, conserved within either PcaD or CatD, may be functionally incompatible within recombinant enzymes. Consequently, selection for avoidance of genetic exchange between pcaD and catD could account for the thorough nt substitution in regions where identical aa were not selected. Sequence repetitions within pcaD suggest that the multiple mutations required for its extensive divergence from catD were achieved in part by acquisition of a complex DNA slippage structure.

Inclusion complexes of the natural product gossypol

Abstract Gossypol, a biologically active compound with a wide spectrum of action (antiviral, immunosuppressive, antifertile, etc.), is a unique compound in the sense of inclusion complex formation. It gives clathrates with all 110 tested low-molecular organic substances. For 80 clathrates single cyrstals have been obtained and their crystallographic parameters have been determined. By the method of X-ray diffraction the structures of 30 inclusion complexes has been solved. The extreme divergence of gossypol clathrate structures is established: clathrates (H-clathrates) with cavities as isolated cells (cryptates), channels (tubulates), intersecting channels (intercalates), clathrates with mixed host-guest matrix and autoclathrates are observed. The existence of 20 groups of isostructural gossypol clathrates is found.

Role of adsorbate interactions in surface dynamics and phase transformations: Mean-field and quasichemical approximation approaches

The significant influence of adsorbate interactions in surface dynamics is quantified using mean-field approximation (MFA) and quasichemical approximation (QCA) approaches and two typical situations (i) T≳Tc (critical temperature for surface phase transformation) and (ii) T<Tc are analyzed. The formulation involves transition state theory (TST) and the key parameters involved are: (1) the sign and magnitude of the pairwise adsorbate interaction energy (w≳0, w<0 meaning repulsive and attractive interactions, respectively) (2) wA♯, the interaction energy between a molecule in the ground state and the activated complex. wA♯A is in turn related to w by a coupling parameter σ. σ=0, σ=1 are shown to result in extreme divergence of the rate behavior for both repulsive and attractive interactions. First T≳Tc is considered. For σ=0, attractive interactions retard and repulsive interactions enhance the surface rates. The rates display nonmonotonic behavior for attractive interactions and steady increase with surface coverage for repulsive interactions. However, when σ=1, the rates monotonically increase for both types of forces. In addition the attractive forces show an instability of the slope due to a cooperative catalytic effect. Both attractive and repulsive forces display maxima when plotted against temperature, the maxima being sharper for the former case. The case T<Tc is more interesting, as a discontinuous phase separation can occur for attractive interactions. The density and internal energy differences between the coexisting phases are computed proceeding from closed-form expressions of the canonical ensemble partition functions and employing standard methods of statistical mechanics. Since repulsive forces can only show continuous order–disorder transitions, they are not considered for T<Tc. The surface rate expressions (both corrected and uncorrected for ground-state internal energy differences between the phases) display a symmetric rate curve (symmetric about θ=0.5) vs surface coverage with a maximum at θ=0.5. A certain type of hole-particle symmetry is present in the rate expression as the rate expression is invariant with respect to the exchange of an occupied and vacant site. This conclusion is valid for both σ=0, σ=1. The appearance of symmetry in the rate curve is suggestive of the phase separation. The qualitative differences between the rate predictions of MFA and QCA are significant enough to warrant refinement in the analysis of surface dynamics.

RNase Protection Analyses Show High Genetic Diversity among Field Isolates of Satellite Tobacco Mosaic Virus

Fifteen field isolates of satellite tobacco mosaic virus (STMV) were collected from wild Nicotiana glauca plants within a 2O-km2 area in southern California. Analyses of the entire length of the satellite genomic RNA of each isolate by RNase protection assays showed that there were 10 different genotypes within the 15 isotates, including some with regions of extreme divergence from the STMV type strain. This indicates a very high level of genetic diversity between natural STMV populations. Isolates from adjacent plants (within 3 m) were not always identical, but were more similar than those from more distant locations. Mapping of the genomic sites of heterogeneity revealed that in all cases the 3′ half of the genome, which comprises the untranslated region, was more conserved than were the 5′ coding regions.

Molecular analysis of the gene encoding a rice starch branching enzyme

The sequence of a rice gene encoding a starch branching enzyme (sbe1) shows extreme divergence from that of the rice gene, that is homologous to bacterial glycogen branching enzyme (sbe2). sbe1 is expressed abundantly and specifically in developing seeds and maximally in the middle stages of seed development. This expression pattern completely coincides with that of the waxy gene, which encodes a granule-bound starch synthase. Three G-box motifs and consensus promoter sequences are present in the 5' flanking region of sbe1. It encodes a putative transit peptide, which is required for transport into the amyloplast. A 2.2 kb intron (intron 2) precedes the border between the regions encoding the transit peptide and the mature protein, and contains a high G/C content with several repeated sequences in its 5' half. Although only a single copy of sbe1 is present in the rice genome, Southern analysis using intron 2 as a probe indicates the presence of several homologous sequences in the rice genome, suggesting that this large intron and also the transit peptide coding region may be acquired from another portion of the genome by duplication and insertion of the sequence into the gene.

The correct sequence of the porcine group C/Cowden rotavirus major inner capsid protein shows close homology with human isolates from Brazil and the U.K.

Amino acid sequence alignments between the human group C/Bristol and the published porcine group C/Cowden VP6 proteins have revealed a region of extreme sequence divergence. We have been unable to confirm the nucleotide sequence of the Cowden VP6 gene corresponding to this region of divergence. Direct sequencing of a PCR-amplified cDNA pool has revealed a frame shift, and three nucleotide changes, within the published sequence of the porcine (Cowden) VP6 gene. The corrected sequence of the porcine protein revealed a closer homology with VP6 from the Bristol strain and two new human group C rotavirus isolates. Atypical rotaviruses have been detected in the feces of children living in Belém, Brazil, and Preston, U.K. Direct sequencing of PCR-amplified cDNA corresponding to the VP6 gene of one isolate from each location confirmed the presence of a group C rotavirus. The complete nucleotide sequences of the VP6 genes from the group C/Belém and C/Preston rotaviruses contained an open reading frame of 1185 nucleotides (395 amino acids; deduced Mr 44,669 Dal. The Belém, VP6 gene demonstrated 97.9% nucleotide homology with the human group C/Bristol VP6 gene and 83.4% nucleotide homology (91.6% deduced amino acid homology) with the corrected porcine group C/Cowden sequence. The Preston VP6 gene demonstrated 99.6% nucleotide homology with the human group C/Bristol VP6 gene and 84.0% nucleotide homology (91.6% deduced amino acid homology) with the corrected porcine group C/Cowden sequence. Remarkably, the deduced amino acid sequence of the Brazilian strain was identical to that of the U.K. isolates.

Surface emitting semiconductor laserdiodes based on surface mode emission

We present novel surface emitting semiconductor laserdiodes based on the excitation and emission of surface modes. By coupling the laser mode to a surface mode we achieve surface emission from conventional laserdiodes with extreme narrow beam divergence. In addition we demonstrate a steering of the beam emission angle by changing the wavelength of the laser light.

Extreme divergence of a novel wheat thionin generated by a mutational burst specifically affecting the mature protein domain of the precursor

A new type of neutral thionin (type V), specifically expressed in developing wheat endosperm, has been found to be encoded by a set of single-copy genes located in the long arms of chromosomes 1A, 1B and 1D, within less than 10,000 base-pairs of those corresponding to the highly basic type-I thionins. Divergence between types I and V has occurred through a process of accelerated evolution that has affected the amino acid sequence of the mature thionin but not the precursor domains corresponding to the N-terminal signal peptide and the long C-terminal acidic peptide. This process involved a deletion and a non-synonymous nucleotide substitution rate equal to the synonymous rate in the thionin sequence.

Genetics ofPotamopyrgus antipodarum(Gastropoda: Prosobranchia): Variation in unisexual populations

Abstract The New Zealand gastropod Potamopyrgus antipodarum comprises both unisexual and bisexual populations. The aim of this study was to examine the genetic variation of unisexual populations and to determine whether the variation observed was consistent with the predictions of ameiotic or meiotic parthenogenesis. Genetic variation within and among eight unisexual populations of P. antipodarum (four from the Auckland region and four from the Rotorua region) was examined for seven presumptive loci. The results indicated extreme genetic divergence both within populations, among populations within each region, and between regions. In many instances a significant excess of heterozy gotes was noted, suggesting ameiotic parthenogenesis. The finding of genetic divergence within individual populations is not consistent with the one-clone one-population hypothesis generally postulated for ameiotic parthenogens. Possible origins of this variation are discussed.

Methodology for measuring hunger and food needs using operant conditioning in the pig

Two reward schedules were compared for their sensitivity in measuring motivational changes due to food restriction in the pig ( Sus scrofa). Six boars were each restricted (proportionally) to 0.8, 0.6 and 0.4 of their predicted ad libitum food intake (PFI) in a Latin square design. Subsequently, they were offered their PFI (Treatment 1.0). Feeding motivation was measured using a fixed ratio of 10 panel presses for each 6-g reward of food (FR schedule) and operant response rates were measured over 20-min sessions at three times post-feeding on each food level. Another 6 boars were subjected to identical conditions, differing only in their being rewarded on a progressive ratio (PR) where the response contingency was incremented by one on each successive reward. Reward rate per session was strongly affected by food restriction on both FR and PR schedules. On FR, reward rate per session increased with food restriction, but only up to a maximum on Treatment 0.6. On PR, in contrast, it was possible to distinguish between reward rates on Treatments 0.6 and 0.4 up to 5 h post-feeding. Progressive ratio appears to be a more sensitive means that FR of measuring changes in feeding motivation, however the cost was found to be an increase in the variability of the response data. The results suggest that the subjects on Treatment 0.6 were maximally food motivated for at least 19 h of the day. This also applies to sows and boars maintained on similar levels of food restriction under commercial conditions, showing the extreme divergence between food-restricted pigs' motivational need for food and their economically determined food allowances.

Chronology and correlation of Quaternary magnetostratigraphy and nannofossil biostratigraphy in Norwegian-Greenland Sea sediments

Paleomagnetic analyses of a series of sediment cores from the Norwegian—Greenland Sea document the existence of seven short-lived geomagnetic events during the last about 500.000 years. Calcareous nannofossil biostratigraphy, correlated to oxygen isotope chronostratigraphy, provides a time framework to infer age and duration of these periods of extreme divergence of the earth's magnetic paleofield from the regular normal polarity configuration of the Brunhes Chron. The geomagnetic events typically extend over 5 to 10 kyr. Two events are observed within the oxygen isotope stage 2–4 interval. A short event is recorded at the stage 4/5 boundary and another just below the stage 5/6 boundary. Additional three geomagnetic events are identified in oxygen isotope stage 6, at the stage 9/10 boundary and near the top of oxygen isotope stage 13, respectively.

Determination of copy number and linkage relationships among five actin gene subfamilies in Petunia hybrida

The actin gene superfamily of Petunia hybrida cv. Mitchell contains greater than 100 gene members which have been divided into several highly divergent subfamilies [1]. Five subfamily-specific probes have been used to compare the actin genes among the Mitchell, Violet 23 (V23) and Red 51 (R51) cultivars of P. hybrida. The sum total of actin genes in these five subfamilies was estimated to be between 10 and 34 members in both V23 and R51. Restriction fragment length polymorphisms (RFLPs) between V23 and R51 were examined with these five probes and eleven different restriction endonucleases. Among the 55 comparisons, 87% exhibited RFLPs. These data indicate extreme divergence between V23 and R51 in DNA sequence and/or the presence of small insertions and deletions surrounding these actin gene subfamilies. This divergence suggests that V23 and R51, which have contrasting phenotypic marker loci on every chromosome, may be useful for the development of a complete RFLP linkage map of the Petunia genome. The segregation of Hind III RFLPs among the progeny of two backcrosses demonstrated that representatives of the five subfamilies of Petunia actin genes exist at four distinct genetic locations and suggested that two of these loci are tightly linked. Apparently, amplification of the numerous members of the Petunia actin gene superfamily occurred via gene dispersal of the original subfamily progenitors and not primarily as a result of amplification of a single chromosomal region.

Lessing’s Tragic Topography: The Rejection of Society and its Spatial Metaphor in Philotas

In exploring Lessing’s use of stage space for illumination of seemingly similar conflicts in his tragedies, the extreme divergence of Philotas from Mis Sara Sampson and Emilia Galotti becomes apparent. The spatial metaphor in Philotas reveals the protagonist’s determined isolation of himself from the social realm and ultimately leads to an understanding of Lessing’s implicit condemnation of Philotas’ radical interiority.

Functional and pseudogenes are similarly organized and may equally contribute to the extensive antibody diversity of the IgVHII family.

Eleven germ-line immunoglobulin VH genes have been isolated from a BALB/c genomic library, using a cDNA probe specific for the GAT/NPa variable region. Restriction fragments of all genes were sequenced: two over 800 bp, covering signals of the 5'- and 3'-non-coding regions, three encompassing the complete coding region and part of the 5', the remaining sequences covering most of the V coding region. All sequences pertained to the VHII family, and were compared with the other 13 homologous genes already published. Characteristic features defining the family are clearly visible all along the sequences analyzed, including the 5'-non-coding region, the leader fragment and the intron organization. About half of the compared genes have pseudogene characteristics, defined either by a stop codon in the coding region or the lack of an initiator codon in the leader segment. Analysis of the replacement mutations, as compared with silent ones, indicate that they are highly clustered in complementarity determining regions, for both the functional and the pseudogenes, suggesting that all genes have been submitted to similar selective pressure, and that the pseudogene repertoire may be actively used, by recombination and/or conversion process. Signals that regulate transcription are highly conserved through the family barriers. The VHII group is the largest Ig V genes family, with extreme sequence divergences reaching 22% nucleotide differences. As no two genes were found identical out of the 24 members compared, and as two genes were found to differ by as little as three nucleotides, it seems that the previous estimate of 60 members might be much too low.

Morphology of identified interneurons in the CA1 regions of guinea pig hippocampus.

Identified interneurons in the CA1 region of guinea pig hippocampus were examined using light and electron microscopic (EM) techniques. The HRP was intracellularly injected into cells, recorded in the in vitro slice preparation, which met physiological criteria for interneurons. These neurons were characterized at the light and electron microscopic level, and used as standards for identifying interneurons which had not been HRP-labeled. Pyramidal basket cell somata were found in stratum oriens and stratum pyramidale; they were easily identifiable by their convoluted nucleus, dense endoplasmic reticulum, and numerous organelles. Aspinous dendrites reached into stratum oriens, where they received profuse synaptic input (primarily asymmetric synapses). Many of the asymmetric synapses degenerated following commissural lesions, suggesting that much of the input to interneurons was from extrinsic afferents. Dendrites were characterized by their spindled appearance, especially at distal sites. They showed postsynaptic degenerative changes following commissural lesions. Interneuron axons were extremely fine, with regular enlargements or "beads" which made apparent synaptic contacts primarily on pyramidal cell somata. The axon of a single, HRP-injected interneuron made many apparent contacts on large numbers of pyramidal cells; axons arborized over distances of several hundred micra within stratum pyramidale. This study provides direct evidence that neurons, with an identified inhibitory interneuron function in hippocampus, can mediate feed-forward as well as feed-back (recurrent) inhibition. Interneuron output showed extreme divergence, with influence over large distances. The high density of intracellular organelles in these cells suggested high metabolic activity and demand, perhaps making these interneurons exceptionally vulnerable to trauma-induced damage.

How Many Words Are There in Printed School English?

THE PURPOSE of this research was to determine the number of distinct words in printed school English. A detailed analysis was done of a 7,260 sample from the Carroll, Davies and Richman, Word Frequency Book. Projecting from this sample to the total vocabulary of school English, our best estimate is that there are about 88,500 distinct words. Furthermore, for every a child learns, we estimate that there are an average of one to three additional related words that should also be understandable to the child, the exact number depending on how well the child is able to utilize context and morphology to induce meanings. Based on our analysis, a reconcilation of estimates of children's vocabulary size was undertaken, which showed that the extreme divergence in estimates is due mainly to the definition of word adopted. Our findings indicate that even the most ruthlessly systematic direct vocabulary instruction could neither account for a significant proportion of all the words children actually learn, nor cover more than a modest proportion of the words they will encounter in school reading materials.

Testing the generic structure revealed by numerical methods in the family Phytoseiidae (Acarina: Mesostigmata)

There has been extreme divergence of opinion about the generic structure of the family Phytoseiidae. An objective evaluation of this structure, obtained by numerical taxonomy, was tested by the method of creating a neutral model based on species distributed at random in morphological space and sampled from hyperellipsoids having the same boundaries as the postulated genera. The numerical taxonomic solution is substantially supported by this test. Minor subjective taxonomic revisions of the numerical solution showed improvement of fit, indicating that the taxonomist's judgment remains an important part of the process of classification.

The nucleotide sequences of several tRNA genes from rat mitochondria: common features and relatedness to homologous species.

We have determined the nucleotide sequences of thirteen rat mt tRNA genes. The features of the primary and secondary structures of these tRNAs show that those for Gln, Ser, and f-Met resemble, while those for Lys, Cys, and Trp depart strikingly from the universal type. The remainder are slightly abnormal. Among many mammalian mt DNA sequences, those of mt tRNA genes are highly conserved, thus suggesting for those genes an additional, perhaps regulatory, function. A simple evolutionary relationship between the tRNAs of animal mitochondria and those of eukaryotic cytoplasm, of lower eukaryotic mitochondria or of prokaryotes, is not evident owing to the extreme divergence of the tRNA sequences in the two groups. However, a slightly higher homology does exist between a few animal mt tRNAs and those from prokaryotes or from lower eukaryotic mitochondria.