Linkage group IV of fish of the genus Xiphophorus (Poeciliidae): Assignment of loci coding for pyruvate kinase-1, glucosephosphate isomerase-1, and isocitrate dehydrogenase-1

Electrophoretic variation ascribable to three enzyme loci, coding for a pyruvate kinase (PK1), a glucose phosphate isomerase (GPI1), and an isocitrate dehydrogenase (IDH1), was observed in three species of fish of the genus Xiphophorus. Electrophoretic patterns in F₁ hybrid heterozygotes confirmed the dimeric structures of GPI and IDH, and indicated a multimeric structure for pyruvate kinase. Variant alleles at the three loci exhibited normal Mendelian segregation in backcross hybrids. Linkage analyses indicate a gene order and estimated recombination of PK1—10%—GPI1—41%—IDH1. No significant interference or sex- or population-specific recombination difference was detected. This group (designated linkage group IV) was shown to assort independently from the nine loci comprising linkage groups I, II, and III and from 23 other informative markers, within the limits of the data. No conclusions with respect to homology of linkage relationships could be reached, due to the presence of presumably duplicated loci in these fish coding for isozymes whose homology with enzymes in other vertebrate species is as yet unestablished.This work was supported in part by Public Health Service Research Grant CA-28909.     

Linkage of two enzyme loci in the fish genus Poecilia (Teleostei:Poeciliidae).

The linkage of loci coding for glucose-6-phosphate dehydrogenase (G6PD) and phosphogluconate dehydrogenase (PGD) is described in fish of the genus Poecilia (Teleostei:Poeciliidae) and designated Poecilia linkage group I. These two loci were shown to assort independently from six other informative markers (peptidase S, malate dehydrogenase 2 [soluble], mannose phosphate isomerase, parvalbumin 2, phosphoglucomutase, and glyceraldehyde-3-phosphate dehydrogenase 2) within the limits of the data obtained. Data for the linkage analyses were generated by scoring starch-gel electrophoretic phenotypes of the eight loci in reciprocal backcross hybrids obtained from matings between Poecilia perugiae and P. vittata. The linkage chi 2 for G6PD-PGD locus pairs was significant (P less than 0.001) in all reciprocal backcross hybrid broods (22.7% recombinants in the combined data), indicating linkage in both parental species. The linkage of G6PD and PGD in gene maps of the poeciliid genera Xiphophorus and Poeciliopsis documents homology of this linkage within the family. Linkages in salmonid and centrarchid fishes suggest conservation of this linkage group in most or all teleosts. The six additional indpendently assorting loci have been assigned to independent linkage groups in Xiphophorus; thus, no example of poeciliid linkage group divergence has yet been identified.     

Linkage group VI of fish of the genus Xiphophorus (Poeciliidae): Assignment of genes coding for glutamine synthetase,uridine monophosphate kinase,and transferrin

Electrophoretic variation ascribable to three protein-coding loci, coding for glutamine synthetase (GS), uridine monophosphate kinase (UMPK), and transferrin (Tf), was observed in three species of fish of the genus Xiphophorus. Electrophoretic patterns in interspecific F1 hybrid heterozygotes suggested monomeric subunit structures of UMPK and Tf and a multimeric structure of undetermined subunit number of GS. Linkage analyses in backcross hybrids indicated a recombination map of GS-0%-Tf-10.8%-UMPK. This group (designated Xiphophorus linkage group VI) was shown to assort independently from the 14 enzyme loci assigned to linkage groups I-V and from 19 other informative markers within the limits of the data.     

Genetic linkage relationships between two enzyme loci,Est-2 and acph,in the mosquito Aedes (Finlaya) togoi

An esterase locus (Est-2), coding for carboxylesterase, and an acid phosphatase locus (Acph) were genetically studied by agar gel electrophoresis in the mosquito Aedes (Finlaya) togoi. The Est-2 and Acph variants occur as a monomer and a dimer, respectively. Both enzyme loci are linked to the sex locus (M) and s (straw-colored larva); the gene arrangement and recombination distances were Est-2—12.6%—s—31.7%—M—2.9%—Acph—3.2%—Est-3. The Est-3 locus was previously shown to code for carboxylesterase.This work was supported by Grant AI 16983-02 from the National Institutes of Health, Bethesda, Md.     

Regional assignment of ADA and ITPA to mouse chromosome 2 (C1----ter). A demonstration of the conserved linkage of enzyme and proto-oncogene loci

Similarity of G-band patterns between the long arm of Chinese hamster chromosome 6 and mouse chromosome 2, combined with the assignments of AK1, ADA, and ITPA to hamster chromosome 6 and AK1 to mouse chromosome 2, suggested mouse chromosome 2 also might contain ADA and ITPA. Here, concordant segregation analysis of enzyme loci and chromosomes in mouse spleen X CHO as well as mouse microcell X CHO somatic cell hybrids established the assignments of ADA and ITPA onto mouse chromosome 2 in the region between the first G-band and the terminus (C1----ter). This assignment presents a demonstration of the conservation and evolution of enzyme and proto-oncogene loci linkage since two cellular homologs of viral oncogenes--c-src and c-abl--also map to mouse chromosome 2. In humans c-src, ADA, and ITPA remain conserved on chromosome 20, whereas AK1 and c-abl are together on chromosome 9. These observations and concepts are discussed with respect to the role of proto-oncogenes in chromosomal evolution and suggest the long arm of chromosome 6 as a fruitful place to look for c-src and c-abl in the Chinese hamster.     

Linkage assignment of a DNA sequence (ERCC2L1) homologous to a human DNA repair gene in Xiphophorus fishes: implications for the evolutionary derivation of human chromosome 19.

Fish gene mapping studies have identified several syntenic groups showing conservation over more than 400 million years of vertebrate evolution. In particular, Xiphophorus linkage group IV has been identified as a homolog of human chromosomes 15 and 19. During mammalian evolution, loci coding for glucosephosphate isomerase, peptidase D, muscle creatine kinase, and several DNA repair genes (ERCC1, ERCC2, and XRCC1) appear as a conserved syntenic group on human chromosome 19. When X. clemenciae and X. milleri PstI endonuclease-digested genomic DNA was used in Southern analysis with a human ERCC2 DNA repair gene probe, a strongly cross-hybridizing restriction fragment length polymorphism was observed. Backcrosses to X. clemenciae from X. milleri x X. clemenciae F1 hybrids allowed tests for linkage of the ERCC2-like polymorphism to markers covering a large proportion of the genome. Statistically significant evidence for linkage was found only for ERCC2L1 and CKM (muscle creatine kinase), with a total of 41 parents and 2 recombinants (4.7% recombination, chi 2 = 35.37, P less than 0.001); no evidence for linkage to GPI and PEPD in linkage group IV was detected. The human chromosome 19 synteny of ERCC2 and CKM thus appears to be conserved in Xiphophorus, while other genes located nearby on human chromosome 19 are in a separate linkage group in this fish. If Xiphophorus gene arrangements prove to be primitive, human chromosome 19 may have arisen from chromosome fusion or translocation events at some point since divergence of mammals and fishes from a common ancestor.     

Marked perinatal lethality and cellular signaling deficits in mice null for the two sphingosine 1-phosphate (S1P) receptors,S1P(2)/LP(B2)/EDG-5 and S1P(3)/LP(B3)/EDG-3

Five cognate G protein-coupled receptors (S1P(1-5)) have been shown to mediate various cellular effects of sphingosine 1-phosphate (S1P). Here we report the generation of mice null for S1P(2) and for both S1P(2) and S1P(3). S1P(2)-null mice were viable and fertile and developed normally. The litter sizes from S1P(2)S1P(3) double-null crosses were remarkably reduced compared with controls, and double-null pups often did not survive through infancy, although double-null survivors lacked any obvious phenotype. Mouse embryonic fibroblasts (MEFs) were examined for the effects of receptor deletions on S1P signaling pathways. Wild-type MEFs were responsive to S1P in activation of Rho and phospholipase C (PLC), intracellular calcium mobilization, and inhibition of forskolin-activated adenylyl cyclase. S1P(2)-null MEFs showed a significant decrease in Rho activation, but no effect on PLC activation, calcium mobilization, or adenylyl cyclase inhibition. Double-null MEFs displayed a complete loss of Rho activation and a significant decrease in PLC activation and calcium mobilization, with no effect on adenylyl cyclase inhibition. These data extend our previous findings on S1P(3)-null mice and indicate preferential coupling of the S1P(2) and S1P(3) receptors to Rho and PLC/Ca(2+) pathways, respectively. Although either receptor subtype supports embryonic development, deletion of both produces marked perinatal lethality, demonstrating an essential role for combined S1P signaling by these receptors.     

Synthesis of (Z)-2-((1H-indazol-3-yl)methylene)-6-[11C]methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one as a new potential PET probe for imaging of the enzyme PIM1

(Z)-2-((1H-Indazol-3-yl)methylene)-6-methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one is a potent and selective proviral integration site in moloney murine leukemia virus kinase 1 (PIM1) inhibitor with an IC₅₀ value of 3 nM. (Z)-2-((1H-Indazol-3-yl)methylene)-6-[¹¹C]methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one, a new potential PET probe for imaging of the enzyme PIM1, was first designed and synthesized in 20–30% decay corrected radiochemical yield and 370–740 GBq/μmol specific activity at end of bombardment (EOB). The synthetic strategy was to prepare a carbon-11-labeled Boc-protected intermediate followed by a quick acidic de-protection.     

Studies in the genus Hypericum L. (Clusiaceae). 1 Section 9. Hypericum sensu lato (part 3): Subsection 1. Hypericum series 2. Senanensia,subsection 2. Erecta and section 9b. Graveolentia

Abstract

Part 4(3) of this monographic series of papers on the genus Hypericum is prefaced by an introduction to the genus and a summary of the aims and methods of the project. This is followed by treatments of the remaining parts of sect. 9. Hypericum sensu stricto and the last segregate section from the original sect. Hypericum, sect. 9b. Graveolentia. Both hitherto untreated parts of the reduced sect. Hypericum are mainly Japanese, but some species extend in distribution as far as Kamchatka, eastern Siberia, central China, and Sabah (Mt. Kinabalu). Sect. Graveolentia is North and Central American. Sect. Hypericum subsect. Hypericum series Senanensia contains seven species from northern Japan and adjacent areas, including H. pibairense (Miyabe & Y. Kimura) N. Robson, stat. nov., H. nakaii subsp. miyabei (Y. Kimura) N. Robson, comb. et stat. nov., H. nakaii subsp. tatewakii (S. Watanabe) N. Robson, comb. et stat. nov. and H. senanense subsp. mutiloides (R. Keller) N. Robson, comb. et stat. nov. Sect. Hypericum subsect. Erecta contains 23 species and one hybrid from Japan, Korea, central China, Taiwan, Luzon, Sabah and Sumatera, including H. kawaranum N. Robson, stat. et nom. nov., H. watanabei N. Robson, stat. et nom. nov., H. kimurae N. Robson, stat. et nom. nov., H. pseudoerectum stat. et nom. nov., H. kitamense (Y. Kimura) N. Robson, stat. nov., H. kurodakeanum N. Robson, stat. et nom. nov., H. furusei N. Robson, sp. nov., H. nuporoense N. Robson, sp. nov. and H. ovalifolium subsp. hisauchii (Y. Kimura) N. Robson, stat. nov. Sect. Graveolentia contains nine species and one hybrid from southeastern Canada, the eastern half of the United States, Mexico and western Guatemala, including H. oaxacanum subsp. veracrucense N. Robson, subsp. nov. and H. macvaughii N. Robson, sp. nov.     

Biosynthesis in vitro of a globoside containing a 2-acetamido-2-deoxy-beta-D-galactopyranosyl group (1----3)-linked and Forssman glycolipid by two N-acetylgalactosaminyltransferases from chemically transformed guinea pig cells

Two N-acetylgalactosaminyltransferase activities (GalNAcT-2 and GalNAcT-3) have been characterized in chemically transformed, cultured guinea-pig cell lines (104C1 and 106B). Line 104C1 is a benz[a]pyrene-transformed tumorigenic variant, whereas line 106B is a 7,12-dimethylbenz[a]anthracene-transformed nontumorigenic variant obtained from fetal guinea-pig cells at 43 days of gestation. The GalNAcT-2 (UDP-GalNAc:GbOse3Cer beta-N-acetylgalactosaminyltransferase) isolated from both 104C1 and 106B cells catalyzed the transfer of Gal-NAc from UDP-GalNAc to the 3H-labeled terminal galactose group of Gb3 [( 6-3H]Gal alpha 1----4Gal beta 1----4Glc----Cer). The 3H-labeled globoside was purified and then subjected to exhaustive methylation. After acetolysis, the partially methylated sugars were separated by two-dimensional, thin-layer chromatography. 3H-Label was detected in two major areas, 2,4,6-tri-O-Me-Gal (40%) and 2,3,4,6-tetra-O-Me-Gal (46%). In a separate experiment, 80% of the GalNAc was released when labeled GbOse4Cer [( 3H]GalNAc----Gal alpha 1----4Gal beta 1----4Glc----Cer) was treated with purified clam beta-hexosaminidase. The present results establish the formation of a beta-D-GalpNAc-(1----3) linkage in the terminal region of the biosynthesized globoside. GalNAcT-3 activity (UDP-GalNAc:GbOse4Cer alpha-GalNAc-transferase), which catalyzes the transfer of GalNAc from UDP-[14C]- or -[3H]GalNAc to GbOse4Cer (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc----Cer), was three times higher in 106B cells than in 104C1 cells. The isolated, purified radioactive product formed an immunoprecipitin line against rabbit anti-Forssman antibody.     

N-alkylated cyclen cobalt(III) complexes of 1-(chloromethyl)-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol DNA alkylating agent as hypoxia-activated prodrugs

A series of cobalt complexes of the potent DNA minor groove alkylator 1-(chloromethyl)-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol (seco-6-azaCBI-TMI) were prepared from a series of N-substituted cyclen ligands. The final N-substituted complexes carried formal overall charges ranging from +2 to -2 and showed limited improvements in solubility. They showed similar stabilities to that of the complex with the unsubstituted cyclen ligand, and large but variable attenuation of the cytotoxicity of the free alkylator (2-30-fold), compared to 150-fold for the unsubstituted ligand. However, they had oxic/hypoxic ratios (2-22-fold) comparable to that of the unsubstituted cyclen complex (5).     

Cellular DNA regions involved in the induction of rat thymic lymphomas (Mlvi-1, Mlvi-2, Mlvi-3, and c-myc) represent independent loci as determined by their chromosomal map location in the rat.

The induction of thymic lymphomas by Moloney murine leukemia virus in the rat is linked to provirus integration in at least four independent cellular DNA regions (Mlvi-1, Mlvi-2, Mlvi-3, and c-myc). Because sequences homologous to at least three of these regions (Mlvi-1, Mlvi-2, and c-myc) map to chromosome 15 in the mouse, the question was raised whether they are closely linked in the rat genome and whether provirus integration in any one of these regions affects the same functional domain in rat DNA. In this study, we identified the chromosomal map location of Mlvi-1, Mlvi-2, and Mlvi-3 in the rat by using mouse-rat somatic cell hybrids that lose the rat chromosomes. The results showed that Mlvi-1 maps similarly to c-myc to chromosome 7, and Mlvi-2 maps to chromosome 2. Mlvi-3 probably maps to chromosome 15. We conclude that Mlvi-1, Mlvi-2, and Mlvi-3 are separate and independent genetic loci. Although Mlvi-1 and c-myc map to the same chromosome, they are not related, as determined by hybridization and restriction endonuclease mapping. The chromosomal map location of Mlvi-1 to chromosome 7 and Mlvi-2 to chromosome 2 is interesting, since chromosomal aberrations involving these two chromosomes are reproducibly observed in rat neoplasias induced by a variety of agents.