The occurrence of chloroplastic and cytosolic isoenzymes of phosphoglycerate kinase in a range of plant species

The chloroplastic and cytosolic isoenzymes of phosphoglycerate kinase (PGK; EC 2.7.2.3) of leaves from 18 of a broad range of 21 vascular plant species were separated by either standard or modified anion-exchange Chromatographic procedures. Immunoprecipitation of the isoenzymes with antisera raised against barley chloroplastic and cytosolic PGK isoenzymes showed that the chloroplastic isoenzymes resemble the chloroplastic isoenzymes of other species more closely than the cytosolic isoenzyme of the same species and vice versa for the cytosolic isoenzymes. Each of the two cyanobacterial species tested, yielded only a single PGK fraction on anion-exchange chromatography and gave no reaction with antisera raised against the barley isoenzymes. The cyanobacteria are presumed to contain only a single PGK which is not closely related to either of the barley PGK isoenzymes. In all of the investigated leaf extracts the catalytic activity of the cytosolic PGK was exceeded by that of the chloroplastic PGK with the ratio for many of the C₃ plants falling within the range 595 to 1585 (cytosolic: chloroplastic). The relative amounts of cytosolic PGK activity appeared to be greater in older leaves, in C₄ and CAM plants and in ferns.Abbreviations CAM crassulacean acid metabolism - pgk phosphoglycerate kinase This work was supported by the Science and Engineering Research Council (grant no. GR/E54504) and also the King's College London Research Strategy Fund.     

Genetic control of triosephosphate isomerase isoenzymes in wheat and rye

Summary The patterns of chloroplastic and cytosolic isoenzymes of triosephosphate isomerase were analysed by immunoblotting in leaves of rye, wheat, and some species of Aegilops or Agropyrum. While rye contained solely one chloroplastic and one cytosolic isoenzyme, wheat had a much more complex pattern which can be explained by the presence of three genomes in 6 x wheats (AABBDD) with distinct triosephosphate isomerase genes that provided different subunit species for the dimeric isoenzyme molecules. The 6 × wheats contained five, the 4 × wheats three, and the 2 × wheats only one chloroplastic isoenzyme band. The isoenzyme patterns were in accordance with a potential origin of one of the three chloroplastic triosephosphate isomerase genes of 6 × wheats from an Aegilops ancestor. The descent of the other two genes was, however, not in accordance with common contentions on the general evolution of cultural wheats. In the reciprocal intergeneric hybrids Secalotricum and Triticale both the chloroplastic and the cytosolic isoenzyme patterns of rye and wheat were biparentally inherited, indicating that both isoenzymes were controlled by nuclear genes. When monitored by immunoblotting the chloroplastic triosephosphate isomerase isoenzymes may provide useful genetic markers.     

GENETIC DIFFERENTIATION IN THE FLORIDA SUBSPECIES OF HELIANTHUS DEBILIS (ASTERACEAE)

Techniques of horizontal starch gel electrophoresis were employed to estimate levels of genetic variation within and genetic differentiation among populations of the three Florida subspecies of Helianthus debilis. The subspecies are H. d. debilis, H. d. vestitus, and H. d. tardiflorus. These taxa are very similar with respect to levels of genetic variability. The average values across all populations for proportion of polymorphic loci (25.5%) and number of alleles per polymorphic locus (2.35) are comparable to those found in other outcrossing plants. The average frequency of heterozygotes per locus (0.05) is lower than that found in other outcrossers. Local populations within each subspecies are genetically very similar. The average genetic distance between local populations is D = 0.010 ± 0.001. Subspecies vestitus and subspecies tardiflorus are also genetically very similar (D = 0.015 ± 0.001). However, subspecies debilis is differentiated from both subspecies vestitus (D = 0.121 ± 0.006) and subspecies tardiflorus (D = 0.103 ± 0.005). This genetic differentiation parallels morphological differentiation. The average genetic distance among all three subspecies is D = 0.080 ± 0.003. A moderate amount of genetic differentiation accompanies the process of subspeciation in Helianthus debilis.     

Appearance of Three Chloroplast Isoenzymes in Dark-grown Pea Plants and Pea Seeds

Activity peaks characteristic of the chloroplastic Calvin cycle enzymes triose-phosphate isomerase, ribose 5-phosphate isomerase, and fructose 1,6-diphosphate aldolase are found in isoelectric focusing patterns of dark-grown pea (Pisum sativum) seedlings and seeds. Apparently, in this higher plant these three chloroplastic isoenzymes can be formed in the absence of light and of chloroplast formation.     

Triosephosphate isomerases and aldolases from light- and dark-grown Euglena gracilis

Euglena gracilis synthesizes two distinct types of triosephosphate isomerase which can be resolved by isoelectric focusing. The more acidic Type A isomerase (pI = 4.4) predominates when cells are grown photoautotrophically and is localized in the chloroplasts. The Type B isoenzyme exhibits a more basic isoelectric pH (pI = 4.8), predominates under heterotrophic growth conditions and is of cytoplasmic origin. The two isoenzymes exhibit similar molecular weights (56,000–60,000) and catalytic properties but can be distinguished by their pH activity profiles. The situation parallels that of fructose diphosphate aldolase where a chloroplastic Class I enzyme (pI = 4.6, M_r 120,000) found in autotrophically grown cells can be resolved from the cytoplasmic Class II (pI = 5.7, M_r 88,000) enzyme which predominates under heterotrophic conditions. Inhibition of chloroplastic 70S ribosomal synthesis by chloramphenicol blocks the formation of the Type A triosephosphate isomerase and the Class I aldolase.     

The Activity and Isoform Complement of Glutamine Synthetase in Panicum Species Differing in Photosynthetic Pathway

Anion exchange chromatography and immunoprecipitation have been used to demonstrate the presence of two forms (GS₁, and GS₂) of glutamine synthetase in the leaves of nine species of Panicum representative of C₃, C₄ and C₃-C₄ intermediate-type photosynthesis. GS₂ from the Panicum species, P. miliaceum and P. maximum was more thermostable than GS₁, GS₁, and GS₂ from P. laxum were equally thermostable but GS₂ from all the Panicum species examined was more sensitive to inhibition by N-ethylmaleimide than GS₁. GS₁, and GS₂ were characterised as being cytoplasmic and chloroplastic isoforms respectively by their reaction with N-ethylmaleimide and by immunoprecipitation with antibodies raised against the cytosolic isoform in barley and the chloroplastic form in tobacco. C₃ species were found to have higher activity of the chloroplastic isoform of glutamine synthetase than C₄ species. C₃-C₄ intermediate species had total leaf glutamine synthetase activities similar to those in C₃ species but were found to have a lower chloroplastic isoform content. The results are consistent with the reassimilation of photorespiratory ammonia by chloroplastic glutamine synthetase.     

Isolation and characterization of the cytosolic and chloroplastic 3-phosphoglycerate kinase from spinach leaves

The cytosol and chloroplast 3-phosphoglycerate kinases (3-PGK) from spinach (Spinacia oleracea L.) were purifled to apparent homogeneity. The procedure included a conventional anion-exchange chromatography on DEAE-cellulose and mainly a series of HPLC columns. The charge differences of the two isoenzymes were so small that separation was only successful by anion-exchange chromatography on a HPLC SynChropak AX 300 column. The portion of the two isoenzmyes in leaf tissue was estimated as 5% and 95%. The major 3-PGK was associated with isolated chloroplasts while the other 3-PGK was only found in the soluble cell fraction. The specific activity of the purified enzymes were in the order of 800 units (per milligram of protein). The molecular weight for the two 3-PGKs under nondenaturing (size exclusion chromatography) and denaturing (SDS-PAGE) conditions were in the order of 40 kilodaltons, with the cytosolic 3-PGK being slightly smaller than the chloroplastic 3-PGK. An antiserum against the chloroplastic 3-PGK showed only 4.6% cross-reaction of the chloroplastic 3-PGK with the cytosolic 3-PGK. The kinetics for glycerate-3-phosphate and MgATP²⁻ were biphasic. The presence of Na₂SO₄ changed the MgATP²⁻ dependence to linearity but not the glycerate-3-phosphate dependence.     

To the knowledge of the leaf beetle genus Cystocnemis (Coleoptera, Chrysomelidae)

Four subspecies of Cystocnemis discoidea are distinguished; two subspecies are described as new to science based on investigation of intraspecific variation in different parts of the species range, and ssp. gebleri was resurrected from synonymy. The ways of speciation in highland-hollow landscapes are discussed. The alpine subspecies C. discoidea oreas ssp. n. has an altibiome disjunction with the nominotypical subspecies. Similarity of the alpine subspecies of C. discoidea to the species from the subgenus Entomomela was found to be the reason of permanent confusion of representatives of these taxa. The subgenus Entomomela is transferred from the genus Oreomela to the genus Cystocnemis. A key to species and subspecies of the genus Cystocnemis is given.     

Notes on the ground beetle Pterostichus togyusanus (Coleoptera: Carabidae): Phylogenetic evidence for species status and new distribution record

A flightless carabid beetle, Pterostichus bellatrix (Tschitschérine, 1895), which is endemic to the Korean Peninsula, had been thought to comprise two subspecies: the nominotypical subspecies distributed in northern and central parts of the peninsula, and the subspecies P. bellatrix togyusanus Park and Kwon, 1996, distributed in the southern part of the peninsula. A recent study upgraded these two subspecies to species level, but no convincing evidence was provided for this taxonomic change. Our comparative morphology of external and male genital characters revealed that these two species are paraphyletic with respect to Pterostichus syleus Kirschenhofer, 1997, which was described from Liaoning Province, China. Thus, separate species status of P. bellatrix and P. togyusanus was confirmed phylogenetically. Although P. togyusanus has only been known from the type locality, Mt. Deogyusan, we newly record this species from Mt. Jirisan, a mountain located south of the known locality. A revised key to species of the Pterostichus opacipennis species group (= the subgenus Koreonialoe (s. str.) Park and Kwon, 1996), which now includes 14 species, is also provided.     

On the taxonomic status of Idiomelas himalayensis Della Beffa (Coleoptera,Carabidae: Harpalini)

Idiomelas himalayensis Della Beffa, 1931 is treated as a subspecies of I. (Egaploa) fulvipes (Erichson, 1843), occurring in northern Pakistan and northeastern India. It is easily distinguished from two other subspecies of this species, the nominotypical one and I. fulvipes indus Kataev, 1997, by the dark coloration of the legs. Comparative characteristics of all the three subspecies of I. fulvipes and data on their distribution are given. Idiomelas fulvipes indus is recorded from Myanmar for the first time. A key to the species and the subspecies of Egaploa Alluaud, 1916 is provided.     

Chromatographic and immunological evidence that chloroplastic and cytosolic pea (Pisum sativum L.) NADP-isocitrate dehydrogenases are distinct isoenzymes

Two NADP-isocitrate dehydrogenase isoenzymes designated as NADP-IDH₁ and NADP-IDH₂ (EC 1.1.1.42) were identified in pea (Pisum sativum) leaf extracts by diethylaminoethylcellulose chromatography. The predominant form was found to be NADP-IDH₁ while NADP-IDH₂ represented only about 4% of the total leaf enzyme activity. These enzymes share few common epitopes as NADP-IDH₂ was poorly recognized by the specific polyclonal antibodies raised against NADP-IDH₁, and as a consequence NADP-IDH₂ does not result from a post-translational modification of NADP-IDH₁. Subcellular fractionation and isolation of chloroplasts through a Percoll gradient, followed by the identification of the associated enzymes, showed that NADP-IDH₁ is restricted to the cytosol and NADP-IDH₂ to the chloroplasts. Compared with the cytosolic isoenzyme, NADP-IDH₂ was more thermolabile and exhibited a lower optimum pH. The data reported in this paper constitute the first report that the chloroplastic NADP-IDH and the cytosolic NADP-IDH are two distinct isoenzymes. The possible functions of the two isoenzymes are discussed.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - NADP-IDH NADP-isocitrate dehydrogenase - NADP-IDH₁ cytosolic NADP-IDH - NADP-IDH₂ chloroplastic NADP-IDH     

Molecular phylogeny of the salmonellae: relationships among Salmonella species and subspecies determined from four housekeeping genes and evidence of lateral gene transfer events

The salmonellae are a diverse group of bacteria within the family Enterobacteriaceae that includes two species, Salmonella enterica and Salmonella bongori. In order to characterize the phylogenetic relationships of the species and subspecies of Salmonella, we analyzed four housekeeping genes, gapA, phoP, mdh and recA, comprising 3,459 bp of nucleotide sequence data for each isolate sequenced. Sixty-one isolates representing the most common serotypes of the seven subspecies of Salmonella enterica and six isolates of Salmonella bongori were included in this study. We present a robust phylogeny of the Salmonella species and subspecies that clearly defines the lineages comprising diphasic and monophasic subspecies. Evidence of intersubspecies lateral gene transfer of the housekeeping gene recA, which has not previously been reported, was obtained.     

Heterogeneity of phosphoglucomutase

A survey of the isoenzyme patterns of phosphoglucomutase (PGM) as demonstrated by starch gel electrophoresis has been undertaken in different species. Seven mammals, two birds, one reptile, two amphibians, four fish, and four invertebrates were studied, and in some cases several tissues were examined. In all cases the predominant enzyme present was a group of electrophoretically related enzymes which are believed to all represent expression of the PGM₁ allele. In some species, examples of other groups of isoenzymes were encountered, presumably representing other genetic loci, corresponding to PGM₂ and PGM₃. These were always less in amount. The PGM from the chicken was unique in the survey in that its mobility changed with storage. A somewhat similar although not identical change could be produced by addition of PCMB to the partially purified enzyme. Both altered enzymes, i.e., that resulting from storage and that produced by addition of PCMB, were more heat labile.Supported by a grant from the National Foundation for Neuromuscular Diseases, and by the Mental Retardation and Human Development Research Program (1 PO1 HD 03773-01).     

An evaluation of the species and subspecies of the genus Salmonella with whole genome sequence data: Proposal of type strains and epithets for novel S. enterica subspecies VII,VIII, IX,X and XI

Species and subspecies within the Salmonella genus have been defined for public health purposes by biochemical properties; however, reference laboratories have increasingly adopted sequence-based, and especially whole genome sequence (WGS), methods for surveillance and routine identification. This leads to potential disparities in subspecies definitions, routine typing, and the ability to detect novel subspecies. A large-scale analysis of WGS data from the routine sequencing of clinical isolates was employed to define and characterise Salmonella subspecies population structure, demonstrating that the Salmonella species and subspecies were genetically distinct, including those previously identified through phylogenetic approaches, namely: S. enterica subspecies londinensis (VII), subspecies brasiliensis (VIII), subspecies hibernicus (IX) and subspecies essexiensis (X). The analysis also identified an additional novel subspecies, reptilium (XI). Further, these analyses indicated that S. enterica subspecies arizonae (IIIa) isolates were divergent from the other S. enterica subspecies, which clustered together and, on the basis of ANI analysis, subspecies IIIa was sufficiently distinct to be classified as a separate species, S. arizonae. Multiple phylogenetic and statistical approaches generated congruent results, suggesting that the proposed species and subspecies structure was sufficiently biologically robust for routine application. Biochemical analyses demonstrated that not all subspecies were distinguishable by these means and that biochemical approaches did not capture the genomic diversity of the genus. We recommend the adoption of standardised genomic definitions of species and subspecies and a genome sequence-based approach to routine typing for the identification and definition of novel subspecies.     

Molecular and Biochemical Characterization of Cytosolic Phosphoglucomutase in Maize : Expression during Development and in Response to Oxygen Deprivation

Phosphoglucomutase (PGM) catalyzes the interconversion of glucose (Glc)-1- and Glc-6-phosphate in the synthesis and consumption of sucrose. We isolated two maize (Zea mays L.) cDNAs that encode PGM with 98.5% identity in their deduced amino acid sequence. Southern-blot analysis with genomic DNA from lines with different Pgm1 and Pgm2 genotypes suggested that the cDNAs encode the two known cytosolic PGM isozymes, PGM1 and PGM2. The cytosolic PGMs of maize are distinct from a plastidic PGM of spinach (Spinacia oleracea). The deduced amino acid sequences of the cytosolic PGMs contain the conserved phosphate-transfer catalytic center and the metal-ion-binding site of known prokaryotic and eukaryotic PGMs. PGM mRNA was detectable by RNA-blot analysis in all tissues and organs examined except silk. A reduction in PGM mRNA accumulation was detected in roots deprived of O₂ for 24 h, along with reduced synthesis of a PGM identified as a 67-kD phosphoprotein on two-dimensional gels. Therefore, PGM is not one of the so-called “anaerobic polypeptides.” Nevertheless, the specific activity of PGM was not significantly affected in roots deprived of O₂ for 24 h. We propose that PGM is a stable protein and that existing levels are sufficient to maintain the flux of Glc-1-phosphate into glycolysis under O₂ deprivation.     

The Maghreb – one more important biodiversity hot spot for tiger beetle fauna (Coleoptera,Carabidae, Cicindelinae) in the Mediterranean region

The tiger beetle fauna of the Maghreb region is one of the richest in the Palaearctic, including 22 species and 5 subspecies and 19% of all Palaearctic species of Cicindelinae. Assembled to their chorotypes, the Maghreb tiger beetles fall into eight different groups that include Maghreb endemics (26% of fauna), Mediterranean (7%), West Mediterranean (40%), North African (4%), Mediterranean-Westturanian (4%), West Palaearctic (4%), Afrotropico-Indo-Mediterranean (4%), and Saharian (11%) species. The Mediterranean Sclerophyl and Atlas Steppe are the Maghreb biogeographical provinces with the highest species richness, while the Sahara Desert has the lowest Cicindelinae diversity. Twenty-five cicindelid species and subspecies (93% of Maghreb fauna) are restricted to only one or two habitat types in lowland areas. Only Calomera littoralis littoralis and Lophyra flexuosa flexuosa are recognized as eurytopic species and occur in three types of habitat. The highest tiger beetle diversity characterizes salt marshes and river banks (in both cases 11 species and subspecies or 41% of Maghreb fauna). Approximately 85% of all Maghreb tiger beetle species and subspecies are found in habitats potentially endangered by human activity.     

Latitudinal Variation of Allozyme Frequencies in Populations of Two Freshwater Isopods in Western Europe

Populations of the freshwater isopods, Proasellus meridianus Racovitza and P. coxalis Dollfus were found to be polymorphic at loci coding for the enzymes glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM). PGM allele frequencies in populations of both species were significantly associated with latitude as has been previously reported for the closely related Asellus aquaticus (L.). Co-variation of PGM allele frequencies was also found in sympatric populations of P. meridianus and A. aquaticus. Significant interpopulation variation in GPI allele frequencies occurred in P. coxalis but not P. meridianus, however, this variation was not associated with latitude.     

A review of flying fishes of the subgenus Hirundichthys (Genus Hirundichthys, Exocoetidae). Part 2. Nerito-oceanic species: H. oxycephalus, H. affinis

The second part of the review of the subgenus Hirundichthys s.str. dealt with two nerito-oceanic species of the subgenus which have weakly pronounced “mirror” on the pectoral fins: H. oxycephalus and H. affinis. The validity of H. coromandelensis (Hornell, 1923) as a subspecies of H. oxycephalus is restored. A comparison of local populations showed that H. oxycephalus is a polytypic species and forms three subspecies: nominative H. oxycephalus oxycephalus from the Western Pacific and Eastern Indian Ocean, H. oxycephalus coromandelensis from the Arabian Sea, Bay of Bengal and adjoining waters of the Indian Ocean, and known by the only specimen from the waters of New Guinea H. oxycephalus frereensis ssp.n. Populations of H. affinis from the Western and Eastern Atlantic differ in the color of pectoral fins. Maps showing the geographical distribution of species and subspecies in the World Ocean are drawn up. A key for identification of species and subspecies belonging to the subgenus is proposed.     

Immunochemical comparison of the tyrosinase isoenzymes in some Basidiomycetes

The tyrosinase isoenzymes of six agaric species of Basidiomycetes were separated immunochemically by the agar double-diffusion technique and identified using dopa as substrate. The number of isoenzymes identified varied from ten in Agaricus hortensis to one in Flamula alnicola. The isoenzymes in the other species were identical serologically to corresponding isoenzyme components in the A. hortensis complex, The technique provides a relatively simple means for the analytical comparison of tyrosinase isoenzymes from different organisms.