Multiple fetal and adult hemoglobins in Malaysian Macaca nemestrina: consequences of a-chain variation

The hemoglobins of fetal and adult Macaca nemestrina were investigated electrophoretically and chromatographically. Standard electrophoretic techniques failed to reveal variation in either fetal or adult hemoglobins beyond that previously attributed to the presence of the duplicate γ-chain loci characteristic of this species. However, column chromatography of whole hemolysates on CM-Sephadex or of globin chains on CM-cellulose in 8 M urea revealed that some fetuses had four major hemoglobins and some adults had two major hemoglobin components. All animals examined were of Malaysian origin; the variation described is a consequence of variation in α-chain structure in Malaysian populations of this species, and of the interaction of different α chains with the products of the duplicate γ-chain loci.     

Chloroplast DNA variation in Populus. II. Interspecific restriction fragment polymorphisms and genetic relationships among Populus deltoides,P. nigra,P. maximowiczii,and P. x canadensis

Restriction fragment analysis was conducted to determine interspecific chloroplast DNA (cpDNA) variation and genetic relationships among Populus deltoides, P. nigra, P. x canadensis (P. deltoides x P. nigra), and P. maximowiczii. Total cellular DNAs of these poplars were digested with 16 restriction endonucleases, and Southern blots of the restriction digests were probed with six different cloned cpDNA fragments from Petunia. P. deltoides, P. nigra, and P. maximowiczii each had a distinct chloroplast genome, separated by many restriction-site and restriction-fragment-length mutations, predominantly in the large single-copy region of the genome. P. x canadensis shared the same cpDNA restriction fragment patterns as P. deltoides var. deltoides. P. nigra was most diverged from P. deltoides, and P. deltoides showed close cpDNA relationships to P. maximowiczii. Nucleotide substitutions per site in cpDNA were 0.0036 between P. deltoides and P. maximowiczii, 0.0071 between P. nigra and P. maximowiczii, and 0.0077 between P. deltoides and P. nigra. We suggest that P. nigra should be classified in a new separate section, the Nigrae.     

Genetic characterization and relationships of Populus alba,P. tremula,and P. x canescens,and their clones

Summary Isozyme analysis was conducted on individuals of Populus alba L., P. tremula L., and P. × canescens Smith to genetically characterize and differentiate species, hybrids, and individuals, and to determine genetic relationships among them. Thirty gene loci, with 71 alleles, coding for 15 enzymes were observed. Individuals could be identified on the basis of their multilocus genotypes. There were 21 unique multilocus genotypes among 23 P. alba clones. Five P. alba clones from Canada were genetically distinct from each other. Each of the 18 P. tremula and 15 P. × canescens clones had unique multilocus genotypes. Thirteen clones had a unique genotype at a single locus. Percentage of polymorphic loci, average number of alleles per locus, and mean observed heterozygosity were, respectively, 50.0, 1.86, and 0.085 in P. alba, 51.7, 1.66, and 0.096 in P. tremula, and 51.7, 1.86, and 0.157 in P. × canescens. Populus alba and P. tremula were genetically distinct from each other and could be distinguished by mutually exclusive alleles at Aco-3, P. tremula-specific gene Mdh-3, and allele frequency differences at 6 loci. Populus × canescens had allele contributions of P. alba and P. tremula. However, their allele frequencies were closer to those of P. alba than being truly intermediate. The mean genetic identity was 0.749 between P. alba and P. tremula, 0.987 between P. alba and P. × canescens, and 0.817 between P. tremula and P. × canescens. Canonical discriminant analysis of multilocus genotypes separated P. alba, P. tremula, and P. × canescens into three distinct groups and portrayed similar interspecific relationship as above. Our results suggested that the putative P. × canescens individuals consisted of a mixture of F₁ hybrids of P. alba and P. tremula and their backcrosses to P. alba.Presently with the University of Alberta, and BioGenetica Inc., P.O. Box 8261, Edmonton, Alberta, Canada T6H 4P1     

Comparative population studies of threePieris butterflies,P. rapae,P. melete andP. napi,living in the same area

The adult populations of three Pieris butterflies, P. rapae, P. melete and P. napi, were studied in an area of their coexistence throughout the flight seasons by using the mark-and-recapture method. The study area, about 3×1.5 km, was set up in a farm village surrounded by the mountainous area in Inabu, Aichi Prefecture. The habitats were qualified by the four factors, i. e., oviposition plants, adult nector plants, roosting-sites and light conditions. Between P. rapae and P. napi, there were sharp differences with regards to overall habitat preferences. P. melete had the widest preferences for all the habitat resources, which overlapped greately with requirements of P. rapae and P. napi. P. melete and P. rapae showed similar preferences for oviposition plants, but the former preferred shaded habitats while the latter preferred sunny places. P. melete and P. napi, having similar preferences for shaded situations, showed differences in the preferences for oviposition plants. Moreover, three species of Pieris were different in their preferences for adult nector plants. Thus, they were more likely to partition habitat resources rather than competing for them. The habitat structures of each species in respect of time, space and stability to weather changes were much different each other in the same area. The habitat of P. rapae was temporary, localized and unstable. While, that of P. melete was more permanent, widespread and stable than that of P. rapae. P. napi seemed to live in the intermediate habitat, i. e., permanent, localized and stable one.     

Comparative population studies of threePieris butterflies,P. rapae,P. melete andP. napi,living in the same area

Utilization of patchy habitats by adult populations of three Pieris butterflies, P. rapae, P. melete and P. napi was studied throughout the flight season in an area of their coexistence, about 3×1.5 km, in a farm village in the mountains in Inabu, Aichi Prefecture. Field study was by the mark-recapture method. Results were analyzed by dispersal distances and recapture duration decay curves for adults of different age-classes estimated on the basis of physical condition of their wings, together with supplementary information of daliy egg-laying rate of females, obtained in field cages. Sexually immature, mated femals of P. rapae after teneral stage showed a migratory flight. On the other hand, reproductive females and all males of P. rapae were strongly resident within suitable habitats, and reproductive females begun to lay eggs abundantly at sunny places of newly suitable areas within a short period. P. melete seemed to disperse gradually from emerged stites and females of this species continued to lay some constant numbers of eggs for more than ten days over a wider area. P. napi appeared more like P. melete than P. rapae. The habitats of the three species can be characterized as follows:P. rapae, temporary, continued for pre-reproductive females but localized for reproductive females and all males, and unstable;P. melete, permanent, widespread, and stable;P. napi, permanent, localized, and stable. The numbers of generations of P. rapae, P. melete and P. napi were estimated to be about six, three and three, respectively. Seasonal fluctuations in the number of adults were influenced by the stability of their habitats, i. e., the population size fluctuated sharply in P. rapae, but it was much more stable in P. melete and P. napi. In view of these results, it can be said that P. rapae fits the general characteristics of a r-strategist whereas P. melete and P. napi are more K-strategic than P. rapae.     

Comparison of the Esterases and Acid Phosphatases in Paramecium multimicronucleatum,Syngens 1–5, P. jenningsi,P. caudatum,and the P. aurelia Complex1

ABSTRACT. Forty-eight stocks in Paramecium jenningsi, syngens 1–5 of P. multimicronucleatum, P. caudatum, P. primaurelia, P. biaurelia, and P. tetraurelia were grown axenically and tested for their esterases and acid phosphatases using starch gel electrophoresis. The five esterases and the acid phosphatases previously characterized in species of the P. aurelia complex were also found in P. jenningsi, and three to four of the esterases and the acid phosphatases were found in the P. multimicronucleatum species complex and in P. caudatum. Additional subtypes were observed for each of the enzyme phenotypes in these new (though here unnamed) species of Paramecium. Two of the new acid phosphatase subtypes, which depart radically in mobility and in pattern, were found in syngen 3 of P. multimicronucleatum and in P. caudatum. Except for syngens 1 and 5 in P. multimicronucleatum, the degree of similarity between syngens 1, 5 and 2, 3, and 4 appears to be very low—perhaps even lower than that seen for species in the aurelia complex. More realistically, the syngens of P. multimicronucleatum should be considered as separate species although they are not here given separate taxonomic names. Limited sharing of subtypes occurred between species in different species complexes. This observation suggests that the molecular distances between species complexes may be even greater than between species within a complex.     

Hemoglobins, haptoglobins, and transferrins in indigenous populations of Kenya

Hemoglobin, haptoglobin, and transferrin phenotypes were determined by means of starch-gel electrophoresis for a sample of 226 Kenya hospital patients. Allele frequencies were: Hb_β^S=0.081; Hp¹=0.057; Tf^D=0.038. Hemoglobin S was the only aberrant hemoglobin found in this sample. Transferrins C and D were the only transferrins found. Hemoglobin and transferrin phenotypes were also determined for a sample of 201 newborn Kenya infants. One of these infants had hemoglobins F, S, and C, eight had hemoglobins A, F, and S, and the remainder had hemoglobins A and F. Transferrins C, B, and D were found in this sample. Allele frequencies were: Tf^B=0.008; Tf^D=0.019.     

Studies on the evolutionary relationships between hemoglobins inChironomus pallidivittatus andC. Tentans

Summary The monomeric hemoglobins ofChironomus tentans andC. pallidivittatus have been isolated and separated into their respective components by gel chromatography on Sephadex G-75 and ion-exchange chromatography on DEAE-Sephacel. The amino acid compositions of the purified components are given. The sequence of the 30 N-terminal amino acid residues of one of the monomeric components (Hb I fromC. pallidivittatus) was determined and found to be identical in almost all of its parts with the monomeric hemoglobins ofC. thummi (CTT III and CTT IV).Antibodies against the monomeric hemoglobins Hb I and Hb IIc and the dimeric fraction were highly specific and no cross reaction between dimeric and monomeric hemoglobins could be demonstrated. The antibodies against the monomers crossreact with the monomeric hemoglobins CTT III and CTT IV ofC. thummi. Taken together with genetic data, the immunological results indicate that divergence of monomeric from dimeric forms was an early event in the evolution of the various hemoglobins inChironomus.     

Purification and Characterization of Recombinant Polymeric Hemoglobin P1 of Glycera dibranchiata

The apoprotein of component P1 of the polymeric fraction of the intracellular hemoglobin of the marine polychaete Glycera dibranchiata has been expressed at a high level in Escherichia coli. The expressed globin was reconstituted with heme and purified. The N-terminal sequence of the recombinant P1 is identical to the cDNA-derived sequence of cloned P1 (Zafar et al., Biochem. Biophys. Acta, 1041, 117-123, 1990). Gel filtration, SDS-PAGE, optical spectra over the range 200-650 nm, and circular dichroism over the range 200-250 nm of the purified recombinant P1 were very similar to the polymeric fraction of native Glycera hemoglobin. The molar ellipticity at 222 nm provided an estimate of 77% for the α-helical content of the recombinant P1, in excellent agreement with that calculated from the crystal structure of Glycera monomeric component M-II. Although the oxygen binding affinity of the recombinant P1 is higher than that of the polymeric fraction of Glycera hemoglobin (3-4 torr vs 7-13 torr), which consists of at least six different single-chain hemoglobins, the Hill coefficient is lower (1.0-1.2 vs 1.2-1.4).     

Factors influencing bud break and rooting and mass-scale micropropagation of three Phragmites species: P. karka, P. communis and P. australis

A procedure has been described for the large-scale micropropagation of three Phragmites species, P. karka, P. communis and P. australis, from axillary buds excised from the main and side branches. Position of the buds on the branches had an effect on the bud break and establishment of the cultures under in vitro conditions. Lower buds of P. australis and middle buds of P. karka and P. communis were the most suitable. The presence of yeast extract as one of the ingredients of the sprouting medium helped in the early detection of systemic contamination. Multiple shoot formation and root initiation were obtained on Murashige and Skoog's basal medium supplemented with different concentrations of BA – 0.5 mg/l for P. karka, 0.25 mg/l for P. communis and 0.1 mg/l for P. australis– 0.5 mg/l Kn and 2% sucrose (w/v). Shoots and roots elongated on half-strength MS basal medium with 2% sucrose but without any plant growth regulators. A zone of root hair was observed in the case of P. australis. Hardening occurred on 95% of the plantlets within 30 days of transfer to the polyhouse. Over 10,000 plants were produced from three buds of each species within 9 months. The plants were supplied to a private company for their industrial waste treatment. Received: 1 June 1998 / Revision received: 28 August 1998 / Accepted: 10 October 1998     

Chromosome analysis and DNA homology in threePicea species,P. mariana,P. rubens,andP. glauca (Pinaceae)

A detailed karyotype analysis was made on the somatic complement ofPicea rubens andP. glauca. B-chromosomes were observed in someP. glauca populations. The karyotypes are generally asymmetrical with most of the chromosomes having median to median-submedian centromeres.Picea glauca chromosomes 2, 3, 7, and 8 have secondary constriction on their short arm and chromosome 10 has a secondary constriction on the long arm. Chromosome 3 was the most easily identifiable, as it has two secondary constrictions located on the short arm. InP. rubens, all the chromosomes but chromosomes 8 and 9 have one to four distinctive secondary constrictions. In general, the diagrammatic comparisons show a high degree of similarity amongP. mariana, P. rubens, andP. glauca. GenomicP. mariana probe strongly hybridized to dots of genomic DNA fromP. rubens andP. glauca indicating that there is a high sequence homology among these three species. The synchronizing agent, hydroxyurea was used at different concentrations to enhance the mitotic index of cell suspensions derived from embryogenic cultures. Hydroxyurea at 1.25 mM increased significantly the mitotic index. An increase of hydroxyurea from 1.25 mM to 5 mM and 10 mM resulted in a steady decrease of mitotic index.     

Chloroplast DNA variation in Populus. I. Intraspecific restriction fragment diversity within Populus deltoides,P. nigra and P. maximowiczii

We examined intraspecific chloroplast (cp) DNA variation within Populus deltoides, P. nigra, and P. maximowiczii by restriction fragment analysis using 16 restriction endonucleases and six heterologous probes of cloned Petunia cpDNA fragments. All three Populus species showed intraspecific cpDNA variation, which was intra- and inter-varietal in P. deltoides, intervarietal in P. nigra, and origin-specific in P. maximowiczii. Two varieties of P. deltoides, var deltoides and var occidentalis, showed distinct cp genomes/DNA. Three distinct cp genomes/DNA, separated by a loss or gain of 1 EcoRV restriction site and/or 1 restriction fragment length polymorphism (RFLP), were observed among the individuals of P. deltoides var deltoides. Within P. nigra, cpDNA of var italica was distinct from that of vars nigra and plantierensis by one RFLP and by a loss or gain of one BamHI restriction site. Populus maximowiczii clones of Chinese origin were separated from those of Japanese origin by a gain or loss of one ClaI restriction site in their cpDNA. The estimate of nucleotide substitutions per site in cpDNA was 0.07% between two varieties of P. deltoides, 0.05% between var italica and var nigra or plantierensis of P. nigra, and 0.01% between Japanese and Chinese accessions of P. maximowiczii.     

Assignment of proximal histidyl imidazole exchangeable proton NMR resonances to individual subunits in hemoglobins A,Boston, Iwate and Milwaukee

The proton nmr spectra of the synthetic valency hybrids, α₂(β⁺CN)₂, (α⁺CN)₂β₂ of hemoglobin A and the natural valency hybrids of the mutant hemoglobins Boston, Iwate and Milwaukee have led to the unambiguous assignment of the two proximal histidyl imidazole exchangeable proton signals at 64 and 76 ppm to individual α and β subunits, respectively. New single non-exchangeable proton resonances detected in the extreme downfield region of the spectra of Hbs Boston and Iwate are tentatively assigned to the coordinated tyrosine of the mutated α chains.     

Systematic revision of the genus Phycodrys (Delesseriaceae,Rhodophyta) from New Zealand,with the descriptions of three new species,P. novae-zelandiae sp. nov., P. franiae sp. nov. and P. adamsiae sp. nov.

Two species of Phycodrys, Phycodrys quercifolia (Bory) Skottsberg and Phycodrys profunda E.Y.Dawson were previously recorded from New Zealand. However, an examination of Phycodrys collections from the New Zealand region showed that all were morphologically different from P. quercifolia (Type locality: the Falkland Islands) and P. profunda (Type locality: CA, USA). RbcL sequence analyses established that the New Zealand Phycodrys species formed a natural assemblage within the genus, consisting of three new species: P. novae-zelandiae sp. nov., P. franiae sp. nov. and P. adamsiae sp. nov. Phycodrys novae-zelandiae is the largest of the three, up to 20 cm in height, with a distinct midrib and multicellular, opposite to subopposite lateral macroscopic veins. It has entirely monostromatic blades except near the midrib and veins, and its procarp contains a three-celled sterile group one (st1) and a one-celled sterile group two (st2). Phycodrys franiae was previously treated as a cryptic species among herbarium collections of P. ‘quercifolia’. It is smaller (4–11 cm high) with weakly developed midribs and veins, the blade is tristromatic throughout, except at the growing margins, and the procarp consists of a four-celled st1 and a two–three-celled st2. Phycodrys adamsiae, previously reported as P. profunda, is a small decumbent or prostrate plant, 1–8 cm long, with a midrib and inconspicuous lateral veins. The blades are tristromatic with serrated margins, two–four-celled surface spines and multicellular marginal holdfasts that differ from those of Californian specimens. The tetrasporangia are borne on marginal bladelets. Phylogenetic analyses place the New Zealand species in a separate group that is distantly removed from most other Phycodrys species.     

Interspecific hybridization of phaseolus vulgaris with P. lunatus and P. acutifolius

Summary The influence of genotypic combinations on the growth of hybrid embryos between Phaseolus vulgaris and P. lunatus, and between P. vulgaris and P. acutifolius was examined. All embryos obtained from P. vulgaris × P. lunatus crosses developed only to a stage which appears to be comparable to the pre-heart-shape stage of selfed embryos. Reciprocal crosses were attempted, but pods abscised at a very early stage. Embryos derived from P. vulgaris × P. acutifolius and reciprocal crosses attained the cotyledon stage although no mature seeds were formed. A distinct characteristic of these embryos was the uneven development of the two cotyledons. The rate of growth and final size of these hybrid embryos seemed to be influenced by the genotypes of both parents.Immature embryos were cultured on defined medium and the effects of glutamine and gibberellin (GA₃) were examined. Glutamine was effective in increasing the survival rate; gibberellin had no apparent effect. Plants derived from cultured embryos of P. vulgaris × P. lunatus, P. vulgaris × P. acutifolius and P. acutifolius × P. vulgaris were obtained.Technical paper No. 00 of the Oregon Agricultural Experiment Station. Research was supported by the Oregon Agricultural Experiment Station, the Research Council of Oregon State University (National Institute of Health Biomedical Research Support Grant RR07079) and the Processor Research Council of Oregon. A.R. is supported by an African Graduate Fellowship from the African-American Institute     

Isolation and metabolite production by Penicillium roqueforti,P. paneum and P. crustosum isolated in Canada

Penicillium roqueforti, P. crustosum and P. paneum grow on ensiled grain and recycled feed unless properly treated. The former two species occur also on cut lumber in Canada. These are known to produce a number of secondary metabolites including roquefortine. In cooler dairy production areas, including Scandinavia and North America, cattle toxicosis has been associated with silage contaminated by these fungi. We collected strains associated with cow or cattle toxicoses. The principal metabolites were determined making use of a new extraction method and analysis combining HPLC, LC/MS/MS, and LC/NMR. Penicillium roqueforti and P. crustosum required amino acid nitrogen for metabolite formation and their toxins were formed under conditions of low oxygen (20–30% saturation). Production of roquefortine C occurred on depletion of the available nitrogen and penitrem A on depletion of carbon source. Yield was reduced by excess carbon. Medium osmotic tension (a_w) affected metabolite production by the two species differently. Penicillium paneum was associated with ill-thrift of dairy cows and P. roqueforti was associated with more serious symptoms. Our data suggest a physiological basis for the common occurrence of roquefortine C in silage without serious consequences and the alternative, the presence of roquefortine C and toxicoses. The strain isolated from lumber was the best producer of the toxins studied. This is the first report of the toxigenic potential of P. roqueforti and P. paneum from Canada.     

Somatic embryogenesis and shoot regeneration from intact seedlings of Phaseolus acutifolius A., P. aureus (L.) Wilczek,P. coccineus L., and P. wrightii L.

A rapid, one-step procedure has been developed for inducing direct organogenesis and somatic embryogenesis in cultures of Phaseolus coccineus L., P. acutifolius A., P. aureus L. [Vigna radiata L. Wilczek] and P. wrightii L. Development of somatic embryos and shoot buds occurred within 6–8 weeks of culture from intact seedlings raised on MS (Murashige and Skoog 1962) medium supplemented with N⁶-benzylaminopurine (BAP). Shoot buds or embryoids originated from subepidermal tissue of the regions adjacent to the shoot apex, hypocotyl and cotyledonary axils. While P. acutifolius and P. aureus were regenerated via shoot formation and P. wrightii by somatic embryogenesis, both embryogenesis and shoot regeneration were observed in P. coccineus. Relatively higher levels of BAP, 50–80 M, were found to be optimal for inducing regeneration while lower concentrations were ineffective. About 40–70 shoots and 70–250 somatic embryos were produced per responding seedling. Regenerated shoots and somatic embryos developed into whole plants on a basal medium or the one supplemented with 1 M naphthaleneacetic acid.     

Initiation of somatic embryogenesis in Pinus banksiana, P. strobus, P. pinaster, and P. sylvestris at three laboratories in Canada and France

During 2002–2004, three laboratories in Canada and France collaborated to improve initiation of somatic embryogenesis (SE) in jack pine (Pinus banksiana Lamb.), eastern white pine (P. strobus L.), maritime pine (P. pinaster Ait.), and Scots pine (P.␣sylvestris L.), giving particular attention to the effects of (1) N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) versus various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA), (2) differences in basal nutrient media, i.e., macro- and microelements, and (3) gelling agent concentration. The work was carried out separately at␣each laboratory, but the details of media compositions were shared and tested on their respective species. Results indicate that the developmental stage of the zygotic embryo (ZE) and genotype effects had a large influence on SE initiation, and that genetic effects were consistent over time. Different species responded differently to PGR types and concentration, basal nutrient media, trace elements, and their combinations. Currently, our best initiation rates based on a selected group of genotypes, optimal development stage of ZE, and medium are 3.9% for jack pine, 54.6% for eastern white pine, 76.2% for maritime pine, and 19.7% for Scots pine.     

Regeneration of transgenic Picea glauca, P. Mariana, and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens

Summary Transgenic plants of three Picea species were produced after coculture of embryogenic tissue with the disarmed strain of Agrobacterium tumefaciens C58/pMP90/pBIV10 and selection on medium containing kanamycin. In addition to the nptII selectable gene (conferring resistance to kanamycin), the vector carried the uidA (β-glucuronidase) marker gene. Transformation frequencies were dependent on the species, genotype, and post-cocultivation procedure. Of the three species tested, P. mariana was transformed at the highest frequency, followed by P. glauca and P. abies. The transgenic state of the embryogenic tissue was initially, confirmed by histochemical β-glucuronidase (GUS) assay followed by Southern hybridization. One to over five copies of T-DNA were detected in various transgenic lines analyzed. Transgenic plants were regenerated for all species using modified protocols for maturation and germination of somatic embryos.