Comparison of polymerase insertion and extension kinetics of a series of O2-alkyldeoxythymidine triphosphates and O4-methyldeoxythymidine triphosphate

The effect of alkyl group size on ability to act as deoxythymidine triphosphate (dTTP) has been studied for the carcinogen products O2-methyl-, O2-ethyl-, and O2-isopropyl-dTTP by using three types of nucleic acids as template and DNA polymerase I (Pol I) or Klenow fragment as the polymerizing enzymes. Apparent Km and relative Vmax values were determined in primer extension on M13 DNA at a single defined site, in poly[d(A-T)], and in nicked DNA. These data are the basis for calculation of the relative rate of insertion opposite A, relative to dTTP. The insertion rate for any O2-alkyl-dTTP is much higher than for a mismatch between unmodified dNTPs. Unexpectedly, O2-isopropyl-dTTP is more efficiently utilized than O2-methyl-dTTP or O2-ethyl-dTTP on each of the templates. O2-isopropyl-dTTP also substitutes for dTTP over extended times of DNA synthesis at a rate only slightly lower than that of dTTP. Parallel experiments using O4-methyl-dTTP under the same conditions show that it is incorporated opposite A more frequently than is O2-methyl-dTTP. Therefore, both the ring position and the size of the alkyl group influence polymerase recognition. Once formed, all O2-alkyl-T.A termini permit elongation, as does O4-methyl-T.A. In contrast to the relative difficulty of incorporating the O-alkyl-dTTPs, formation of the following normal base pair (C.G) occurs rapidly when dGTP is present. This indicates that a single O-alkyl-T.A pair does not confer significant structural distortion recognized by Pol I.     

Limited genetic differentiation of Mycetomoellerius mikromelanos in Parque National Soberanía,Panama: Implications for queen dispersal

The coevolutionary relationship between fungus-growing ants (Formicidae: Attini: Attina) and their symbionts has been well studied in the Panamanian rain forests. To further understand the ecological context of these evolutionary relationships, we have examined the population-genetic structure of the fungus-growing ant species Mycetomoellerius mikromelanos Cardenas, Schultz, Adams 2021 in the Panama Canal Zone. We specifically investigated the presence of population structure, the significance of geographic features (i.e., creeks) limiting gene flow, and relatedness between ant colonies. To accomplish this, we genotyped 85 ant colonies from nine creeks across an approximately 30 km transect in Parque National Soberanía, Panama, using double-digest restriction-site-associated DNA sequencing. We did not find distinct population structure using two genetic clustering methods; however, we did detect an effect of isolation by distance. Furthermore, related colonies were frequently detected on the same creek or neighboring creeks, and some at further geographic distances. Collectively, these findings demonstrate that new colonies tend to establish on natal creeks and occasionally on distant creeks following long-distance dispersal events. We discuss how population-genetic patterns reveal the natural history of M. mikromelanos in Parque National Soberanía and how these results fit into the context of fungus-growing ant mutualisms. Abstract in Spanish is available with online material.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Somatic embryogenesis from leaf callus of mature plants of the gymnospermCeratozamia mexicana var. robusta (Miq.) dyer (Cycadales)

Summary Embryogenic callus was induced from explanted pinnae of newly emerged leaves of mature plants ofCeratozamia mexicana var. Robusta (Gymnospermae, Cycadales) on a modified B5 formulation with 1 mg·liter⁻¹ kinetin and 1 mg·liter⁻¹ 2,4-dichlorophenoxyacetic acid. Proembryos developed on induction medium, but they were more numerous after subculture onto phytohormone-free medium, which also enabled suspensors to elongate. For nearly 1.5 yr after explanting, subsequent development of somatic embryos was not observed as suspensors dedifferentiated to form embryogenic callus on phytohormone-free medium. After this time, cotyledonary somatic embryos developed at the distal end of the suspensors. Somatic embryos have germinated on phytohormone-free medium. This is the first report of regeneration by somatic embryogenesis of a gymnosperm species from a mature tree. This technique has great potential for preservation of the highly endangered cycads.     

Resonance Raman investigation of a soluble cytochrome c552 from alkaliphilic Bacillus firmus RAB

The environment of the heme site of a low-potential soluble cytochrome (c552) from alkaliphilic Bacillus firmus RAB has been characterized with resonance Raman scattering and compared to that of horse heart cytochrome c. The Raman data indicate that vibrational bands sensitive to the axial ligation of the heme, as well as modes sensitive to the heme peripheral environment in cytochrome c552, are distinct from those of horse heart cytochrome c. The spectra of cytochrome c552 display resonance Raman modes indicative of a methionine as the sixth ligand in the oxidized form, while the reduced form appears to contain a nitrogenous-based sixth ligand. In addition, Q-band excitation reveals differences among vibrational modes in cytochrome c552 that are sensitive to the amino acid environment surrounding the heme.     

Dynamics and reactivity of HbXL99 alpha. A cross-linked hemoglobin derivative

Resonance Raman spectroscopy, transient absorption, and fluroescence techniques have been employed to investigate the structure and dynamics of the alpha-cross-linked hemoglobin derivative, HbXL99 alpha. The resonance Raman spectra of the deoxy form of HbXL99 alpha are identical to those of native NbA (VFe-His approximately 222 cm-1), which exhibit a T-state (low affinity) structure regardless of solvent conditions. The resonance Raman spectra of the transient heme photoproduct resulting from CO photolysis from HbXL99 alpha appear to have structures intermediate between deoxy-T and ligand-bound R structures (VFe-His approximately 222 cm-1). Time-resolved resonance Raman data of HbXL99 alpha-CO show that complete CO recombination occurs after approximately 5 ms, with only a small amount of the CO-bound species reforming within approximately 200 ns (geminate recombination). Transient absorption spectra of HbXL99 alpha-O2 indicate that the extent of sub-nanosecond geminate recombination of O2 is also reduced in the cross-linked derivative relative to native HbA. The decrease in tryptophan fluorescence of HbXL99 alpha upon oxygenation further indicates that tertiary structural changes at the alpha 1-beta 2 interface upon ligation are apparently reduced, but not eliminated in the cross-linked derivative relative to HbA.     

Long‐term persistence of wildlife populations in a pastoral area

Facilitating coexistence between people and wildlife is a major conservation challenge in East Africa. Some conservation models aim to balance the needs of people and wildlife, but the effectiveness of these models is rarely assessed. Using a case‐study approach, we assessed the ecological performance of a pastoral area in northern Tanzania (Manyara Ranch) and established a long‐term wildlife population monitoring program (carried out intermittently from 2003 to 2008 and regularly from 2011 to 2019) embedded in a distance sampling framework. By comparing density estimates of the road transect‐based long‐term monitoring to estimates derived from systematically distributed transects, we found that the bias associated with nonrandom placement of transects was nonsignificant. Overall, cattle and sheep and goat reached the greatest densities and several wildlife species occurred at densities similar (zebra, wildebeest, waterbuck, Kirk's dik‐dik) or possibly even greater (giraffe, eland, lesser kudu, Grant's gazelle, Thomson's gazelle) than in adjacent national parks in the same ecosystem. Generalized linear mixed models suggested that most wildlife species (8 out of 14) reached greatest densities during the dry season, that wildlife population densities either remained constant or increased over the 17‐year period, and that herbivorous livestock species remained constant, while domestic dog population decreased over time. Cross‐species correlations did not provide evidence for interference competition between grazing or mixed livestock species and wildlife species but indicate possible negative relationships between domestic dog and warthog populations. Overall, wildlife and livestock populations in Manyara Ranch appear to coexist over the 17‐year span. Most likely, this is facilitated by existing connectivity to adjacent protected areas, effective anti‐poaching efforts, spatio‐temporal grazing restrictions, favorable environmental conditions of the ranch, and spatial heterogeneity of surface water and habitats. This long‐term case study illustrates the potential of rangelands to simultaneously support wildlife conservation and human livelihood goals if livestock grazing is restricted in space, time, and numbers.