Phosphoglucomutase electrophoretic variants in the mouse

The phosphoglucomutase (PGM) electrophoretic phenotype of the mouse (Mus musculus) consists of several distinct components which can be grouped into two major zones designated PGM-1 and PGM-2. Evidence presented here indicates that each zone is controlled by a single genetic locus denoted Pgm-1 and Pgm-2, respectively. Two variant forms segregated at the Pgm-1 locus. They were codominantly expressed and inherited as alleles at an autosomal locus. The alleles were termed Pgm-1 ^a (fast) and Pgm-1 ^b (slow). These alleles were separately fixed in a number of inbred strains of mice. Preliminary evidence based on wild mouse phenotypes indicates that variant forms also exist for PGM-2 which are inherited as alleles at an autosomal locus. Genetic linkage relationships have not been determined for these loci. PGM-1 variants and PGM-2 were expressed in mouse fibroblasts in vitro.Supported by U.S. Public Health Service grants GM-09966 and GM-07249 from General Medical Sciences and 5 F2 HD-35,531 from Child Health and Human Development; and Atomic Energy Commission contract AT(30-1)-3671.Postdoctoral Fellow of the U.S. Public Health Service.     

Variation in the mating system of Sitka spruce (Picea sitchensis): evidence for partial assortative mating

Five allozyme polymorphisms were used to analyze the mating system in a Sitka spruce seed orchard in Saanichton, British Columbia. Allelic frequencies differed between the pollen and maternal pools at three of the five loci, with alleles rare in the maternal pool being even rarer in the effective pollen pool. Minor differences in pollen allelic frequencies were observed in the upper vs. lower crown. The multilocus outcrossing rate of the upper crown (t_m = 0.909) exceeded that of the lower crown (t_m = 0.764). Single-locus estimates of the outcrossing rate were significantly heterogeneous, with the lowest estimate of outcrossing, t = 0.773, observed for PGM-2 locus. Analyses of the mating system for the three maternal PGM-2 genotypes revealed heterogeneous pollen allelic frequencies and heterogeneous outcrossing rates, possibly due to assortative mating at this locus.     

Assignment of human transcobalamin II (TC2) to chromosome 22 using somatic cell hybrids and monosomic meningioma cells

Summary Human transcobalamin II (TC2), a vitamin B12 binding serum protein, is synthesized and secreted into the medium by cells growing in vitro. Mouse-man somatic cell hybrids were analyzed in order to map the locus of TC2. The presence of human TC2 in the culture media was correlated with the results of genetic marker and chromosome analysis of the hybrid cells. Chromosome 22 showed 100% concordancy. However, chromosome 6 (90% concordancy) and chromosome 7 (96% concordancy) were not completely excluded. Meningioma cells obtained from patients heterozygous for TC2 showed a concomitant loss of one chromosome 22 and one of the TC2 alleles, strongly supporting the assignment to chromosome 22.     

Assignment of the human ST2 gene to chromosome 2 at q11.2

The humanSt2 locus has been assigned to chromosome 2, using a human ST2 cDNA clone, by a human/rodent somatic cell hybrid mapping panel. TheSt2 locus has also been mapped to chromosome 2811.2, using a human ST2 genomic DNA clone, by in situ hybridization. The locus is very tightly linked to theIl-1r1 locus. Together with the structural similarity of ST2 to IL-1RI, these data suggest functional relationships between these two genes.     

Phosphopentomutase of Bacillus stearothermophilus TH6-2: The Enzyme and Its Gene ppm

Phosphopentomutase catalyzes the transfer of an intramolecular phosphate on ribose or deoxyribose, and is involved in the salvage pathway of nucleoside synthesis. We identified a sequence 5′-upstream of the genes for the nucleoside phosphorylases of Bacillus stearothermophilus as the phosphopentomutase (ppm) gene. The novel gene corresponded to an open reading frame of 1,179 nucleotides that is translated into a putative 393-amino acid protein with a molecular weight of 43,735. The gene product, partially purified from ppm-overexpressing Escherichia coli cells, was judged to be a monomer of a 44-kDa polypeptide. The phosphopentomutase was found to catalyze the phosphotransfer on not only ribose or deoxyribose but also arabinose or dideoxyribose.     

An immunochemical method for the detection of the expression of human gene loci in human-rodent somatic cell hybrids with special reference to the GPI locus

Species-specific antibodies, prepared against unpurified human and Chinese hamster fibroblast extracts, were used to identify the parental origins of enzymes in human-hamster somatic cell hybrids. Results of the detection of the expression of the human glucosephosphate isomerase gene locus (GPI) by electrophoretic and immunochemical techniques were concordant in 17 instances. The human GPI synthesized by fibroblasts derived from skin explants and by somatic cell hybrids retaining the human GPI locus, regardless of whether the human parental cells were lymphocytes or fibroblasts, appeared to be antigenically identical.This work was supported by the Medical Research Council of Canada (Grant MA-4061). Personnel and operating support were provided by The Children's Hospital of Winnipeg Research Foundation, Inc.     

The UPS locus encoding uroporphyrinogen I synthase is located on human chromosome 11

The expression of the UPS locus encoding uroporphyrinogen I synthase has been investigated in human/mouse somatic cell hybrids. Human and mouse uroporphyrinogen I synthase can be readily distinguished by their isoelectric points. In hybrid cells, both human and mouse isozymes are detected. The multiple human uroporphyrinogen I synthase isozymes segregate as a single unit, as expected if they are the products of a single locus. The absence of new heteropolymers in hybrid cells supports the biochemical evidence that the active enzyme is a monomer. The presence of human uroporphyrinogen I synthase in hybrid clones was correlated with the presence of human chromosome 11, or its enzymatic marker, without exception in 44 independent hybrid lines. All other chromosomes could be eliminated as possible locations for this locus, due to their independent segregation. This report represents the first gene assignment for an enzyme in the heme biosynthesis pathway.     

Genetic instability at the opaque-2 locus of maize

Summary A case of genetic variegation discovered at the opaque-2 locus of maize that includes a two-element system with a receptor and regulatory element is described. The somatic mutability depends on the existence of two genetic factors: a responsive allele (with receptor element), o2m(r), and a regulatory element, Bg, that induces mutability of o2m(r). In the absence of Bg, o2m(r) is indistiguishable from the recessive alleles of the O2 locus; in the presence of the regulatory element, o2m(r) mutates giving rise to sectors of flint-like endosperm in an opaque back-ground. The regulatory element Bg may be located independently or at the controlled locus. The genetic properties of the new system, somatic mutability, transposition, existence of different patterns of mutability, are apparently similar to those previously described in maize for the classical systems of controlling elements. In addition, the recovery of the o2 mutability from crosses between spontaneous o2 alleles suggests that transposable genetic elements may be involved in the origin of natural mutability.     

Molecular identification of mammalian phosphopentomutase and glucose-1,6-bisphosphate synthase, two members of the alpha-D-phosphohexomutase family

The molecular identity of mammalian phosphopentomutase has not yet been established unequivocally. That of glucose-1,6-bisphosphate synthase, the enzyme that synthesizes a cofactor for phosphomutases and putative regulator of glycolysis, is completely unknown. In the present work, we have purified phosphopentomutase from human erythrocytes and found it to copurify with a 68-kDa polypeptide that was identified by mass spectrometry as phosphoglucomutase 2 (PGM2), a protein of the alpha-d-phosphohexomutase family and sharing about 20% identity with mammalian phosphoglucomutase 1. Data base searches indicated that vertebrate genomes contained, in addition to PGM2, a homologue (PGM2L1, for PGM2-like 1) sharing about 60% sequence identity with this protein. Both PGM2 and PGM2L1 were overexpressed in Escherichia coli, purified, and their properties were studied. Using catalytic efficiency as a criterion, PGM2 acted more than 10-fold better as a phosphopentomutase (both on deoxyribose 1-phosphate and on ribose 1-phosphate) than as a phosphoglucomutase. PGM2L1 showed only low (<5%) phosphopentomutase and phosphoglucomutase activities compared with PGM2, but was about 5-20-fold better than the latter enzyme in catalyzing the 1,3-bisphosphoglycerate-dependent synthesis of glucose 1,6-bisphosphate and other aldose-bisphosphates. Furthermore, quantitative real-time PCR analysis indicated that PGM2L1 was mainly expressed in brain where glucose-1,6-bisphosphate synthase activity was previously shown to be particularly high. We conclude that mammalian phosphopentomutase and glucose-1,6-bisphosphate synthase correspond to two closely related proteins, PGM2 and PGM2L1, encoded by two genes that separated early in vertebrate evolution.     

Wildlife genetics and disease: allozyme evolution in the wild boar (Sus scrofa) caused by a swine fever epidemy

Enzyme polymorphism at 42 loci was compared before and after a major epidemy of swine fever in wild boars from northern Vosges (France). No change was observed in the 38 monomorphic loci, but allele frequencies at the phosphoglucomutase locus PGM-2* changed significantly. Possible causes for this observation are discussed, and it appears that PGM-2 locus could be a genetic marker of resistance to this viral disease.     

Dietary Effects on Fitness Components at the PGM-1 Locus of TRIBOLIUM CASTANEUM

The pattern of genetic differentiation among experimental populations of the flour beetle Tribolium castaneum suggested the hypothesis that relative fitness of three genotypes at the PGM-1 locus (or other linked loci) depends directly on diet. This hypothesis was tested by measuring several fitness components (developmental time, survival, fecundity, rate of egg cannibalism) on groups of individuals differing at the PGM-1 locus that were reared on three types of flour (wheat, corn and a mixture of wheat, corn, barley and rye). Flour type had large effects on all traits except larval survival to 3 weeks of age. Relative fitnesses of the three genotypes differed significantly for fecundity. Diet was found to significantly influence the relative developmental times of the three genotypes.     

Physiologic target of the Air-1 trans-activator revealed by stable transfection assay

RJ 2.2.5 is a human B cell mutant, derived from Raji cells, which has lost expression of major histocompatibility complex (MHC) class II genes because of a defect in the AIR1 locus function. The MHC class II-positive phenotype can be restored by introducing an active AIR1 locus or its mouse equivalent, Air-1. An example of the latter is the H4 cell hybrid, derived by somatic cell fusion between RJ 2.2.5 and mouse class II-positive spleen cells. H4 contains a single mouse chromosome, autosome 16, in which the Air-1 locus maps, and an entire RJ 2.2.5-derived genome. In the present study we show that the physiologic target of the Air-1 locus product is contained within a limited HLA-DRA promoter sequence of 300 base pairs, encompassing the crucial Y, X, and W cis-acting elements. A plasmid construct, pDRA300neo, containing the HLA-DRA promoter sequence which drives the expression of the neomycin resistance gene, has been stably integrated in the genome of the H4 hybrid. Transfectants selected in the presence of G418 retain mouse chromosome 16 and express the DR genes. On the other hand, transfectants grown in a non-selective medium segregate mouse chromosome 16; this is accompanied by a loss of DRA gene expression and G418 resistance, although pDRA300neo is still integrated in the genome. These results offer scope for using this experimental model to clone the Air-1 gene in a straightforward way. Correspondence to: R. S. Accolla.     

Intervention of somatic mutational events in vivo by a germline defect at the adenine phosphoribosyltransferase locus

Both germline and somatic mutations are known to affect phenotypes of human cells in vivo. In previous studies, we cloned mutant peripheral blood T cells from germline heterozygous humans for adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) deficiency and found that approximately 1.3 × 10^–4 peripheral T cells had undergone in vivo somatic mutations. Loss of heterozygosity (LOH) was the major cause of the mutations at the APRT locus since approximately 80% of the mutant T cell clones exhibited loss of normal alleles. In the present study, we identified three heterozygous individuals for APRT deficiency (representing two separate families), in whom none of the somatic mutant cells exhibited LOH at the APRT locus. The germline mutant APRT alleles of these heterozygotes from two unrelated families had the same gross DNA abnormalities detectable by Southern blotting. None of the germline mutant APRT alleles so far reported had such gross DNA abnormalities. The data suggest that the germline mutation unique to these heterozygous individuals is associated with the abrogation of LOH in somatic cells. The absence of LOH at a different locus has already been reported in vitro in an established cell line but the present study describes the first such event in vivo in human individuals. Received: 10 May 1996     

Assignment of the gene for glyoxylase I to mouse chromosome 17 by somatic cell genetics

Evidence is presented for the assignment of the gene for glyoxylase I to mouse chromosome 17 using mouse × Chinese hamster somatic cell hybrids. GLO I was not expressed concordantly with any known marker enzymes which represented 11 linkage groups. The presence of chromosome 17 and expression of GLO I were concordant in 31/31 clones. GLO I is thus linked to the H-2 histocompatibility locus in the mouse.     

The GAA triplet-repeat is unstable in the context of the human <Emphasis Type="Italic">FXN</Emphasis> locus and displays age-dependent expansions in cerebellum and DRG in a transgenic mouse model

Friedreich ataxia (FRDA) is caused by homozygosity for FXN alleles containing an expanded GAA triplet-repeat (GAA-TR) sequence. Patients have progressive neurodegeneration of the dorsal root ganglia (DRG) and in later stages the cerebellum may be involved. The expanded GAA-TR sequence is unstable in somatic cells in vivo, and although the mechanism of instability remains unknown, we hypothesized that age-dependent and tissue-specific somatic instability may be a determinant of the progressive pathology involving DRG and cerebellum. We show that transgenic mice containing the expanded GAA-TR sequence (190 or 82 triplets) in the context of the human FXN locus show tissue-specific and age-dependent somatic instability that is compatible with this hypothesis. Small pool PCR analysis, which allows quantitative analysis of repeat instability by assaying individual transgenes in vivo, showed age-dependent expansions specifically in the cerebellum and DRG. The (GAA)₁₉₀ allele showed some instability by 2 months, progressed at about 0.3–0.4 triplets per week, resulting in a significant number of expansions by 12 months. Repeat length was found to determine the age of onset of somatic instability, and the rate and magnitude of mutation. Given the low level of cerebellar instability seen by others in multiple transgenic mice with expanded CAG/CTG repeats, our data indicate that somatic instability of the GAA-TR sequence is likely mediated by unique tissue-specific factors. This mouse model will serve as a useful tool to delineate the mechanism(s) of disease-specific somatic instability in FRDA.     

Identification and chromosomal mapping of a third mouse runt-like locus

The Drosophila runt gene, which controls early events in embryogenesis, has been shown to have homologues in human and mouse. The human gene on 21q22 is involved in the t(8;21) associated with acute myeloid leukemia. Two mouse runt-like loci encoding DNA-binding proteins have been identified. We report here the isolation and partial sequence of a molecular clone of a third mouse runt-like locus. By using a panel of somatic cell hybrids and interspecific backcross mice, we map the novel locus to the telomeric region of mouse chromosome 4.     

Linkage of Agt and Actsk-1 to distal mouse Chromosome 8 loci: a new conserved linkage

Angiotensinogen is an ₂ involved in the maintenance of blood pressure and electrolyte balance. We have refined the position of the mouse angiotensinogen locus (Agt) on Chromosome (Chr) 8 and have also confirmed the assignment of the human angiotensinogen locus (AGT) to Chr 1. The segregation of several restriction fragment length variants (RFLVs) was followed in two interspecific backcross sets and in four recombinant inbred (RI) mouse sets. Analysis of the segregation patterns closely linked Agt to Aprt and Emv-2, which places the angiotensionogen locus on the distal end of mouse Chr 8. Additionally, a literature search has revealed that the strain distribution pattern (SDP) for the mouse skeletal -actin locus 1 (Actsk-1, previously Actal, Acta, or Acts) is nearly identical to the SDP for Agt in two RI sets. On the basis of this information we were able to reassign Actsk-1 to mouse Chr 8. By screening a panel of human-mouse somatic cell hybrids, we confirmed that the human angiotensioogen locus lies on Chr 1. This information describes a new region of conserved linkage homology between mouse Chr 8 and human Chr 1. It also defines the end of a large region of conserved linkage homology between mouse Chr 8 and human Chr 16.     

Genetic control of enzyme polymorphisms in the California vole,Microtus californicus

Inheritance of 15 polymorphic isozymes was investigated in captive Microtus californicus. Eleven of the isozymes show patterns consistent with a Mendelian model of inheritance: glycerol-3-phosphate dehydrogenase (GPD), lactate dehydrogenases A and B (LDH-A and LDH-B), malic enzyme 2 (ME-2), isocitrate dehydrogenase 1 (ICD-1), phosphogluconate dehydrogenase (PGD), glutamate-oxaloacetate transaminase 1 (GOT-1) phosphoglucomutase 2 (PGM-2), leucine aminopeptidase (LAP), glucosephosphate isomerase (GPI), and esterase 2 (ES-2, from kidney). four of the isozymes show patterns that cannot be interpreted by a simple genetic model: esterases 1 and 4 (ES-1, ES-4, from hemolysate), esterase 3 (ES-3, from plasma), and protein 1 (PT-1). The following pairs of loci are assorting independently: LAP and PGD, LAP and PGM-2, GOT-1 and PGD, GOT-1 and GPD, LAP and GPD, GPD and PGD, GPI and PGD. Data from one test cross mating indicate that GPD and PGM-2 are loosely linked with recombination about 30%. Additional data are needed to confirm this relationship.This study was conducted while the first author was the recipient of an NIH Traineeship. The Departments of Genetics and Zoology provided financial support for the maintenance of the animal colony. This work was supported in part by an NIH-Biomed grant (3-S05-RR-07006-08S1) to W. Z. Lidicker.