Genetic control and intracellular localization of glutamate oxaloacetic transaminase in maize

Glutamate oxaloacetic transaminase (L-aspertate: 2-oxoglutarate aminotransferase, E.C. 2.6.1.1; GOT) was found to occur in five distinct electrophoretic forms in different tissue extracts from a number of highly inbred strains of Zea mays L. No major qualitative differences were detected in the various tissues examined, and the isozyme patterns did not undergo changes during temporal development of any given inbred strain. Cell fractionation studies showed one isozyme to be associated with the mitochondria (mGOT), another to be exclusively associated with the soluble fraction (sGOT), and a third to be associated with the glyoxysomes (gGOT). The glyoxysomal form occurs as two electrophoretically distinct variants which exist in different inbred strains of maize. The gGOT variants are under the control of two codiminant alleles (Got1A and Got1B) at the Got1 locus (isozyme5, gGOT). The genetic data and gene dosage effects suggest that GOT in maize is functionally a dimer.     

Molecular Markers for the agouti Coat Color Locus of the Mouse

The agouti (a) coat color locus of the mouse acts within the microenvironment of the hair follicle to control the relative amount and distribution of yellow and black pigment in the coat hairs. Over 18 different mutations with complex dominance relationships have been described at this locus. The lethal yellow (Ay) mutation is the top dominant of this series and is uniquely associated with an endogenous provirus, Emv-15, in three highly inbred strains. However, we report here that it is unlikely that the provirus itself causes the Ay-associated alteration in coat color, since one strain of mice (YBR-Ay/a) lacks the provirus but still retains a yellow coat color. Using single-copy mouse DNA sequences from the regions flanking Emv-15 we have detected three patterns of restriction fragment length polymorphisms (RFLPs) within this region that can be used as molecular markers for different agouti locus alleles: a wild-type agouti (A) pattern, a pattern which generally cosegregates with the nonagouti (a) mutation, and a pattern which is specific to Emv-15. We have used these RFLPs and a panel of 28 recombinant inbred mouse strains to determine the genetic linkage of these sequences with the agouti locus and have found complete concordance between the two (95% confidence limit of 0.00 to 3.79 centimorgans). We have also physically mapped these sequences by in situ hybridization to band H1 of chromosome 2, thus directly confirming previous assignments of the location of the agouti locus.     

α-Hydroxyacid Oxidase Genetics in the Mouse: Evidence for Two Genetic Loci and a Tetrameric Subunit Structure for the Liver Isozyme

We have examined a polymorphism for liver GOX in inbred strains of the mouse Mus musculus. Genetic studies demonstrated that the two phenotypes for this enzyme present in BALB/C and NZC parental strains segregated as though they were controlled by codominant alleles at a single autosomal locus (GOX) which mapped closely to the agouti locus in linkage group V. Kidney HAOX activity is invariant in these inbred strains and is encoded by a separate genetic locus designated HAOX. BALB/C x NZC F(1) hybrid mice exhibited three intermediate forms of liver GOX activity, in addition to the parental enzymes, which is consistent with a tetrameric subunit structure.     

Genetics and ontogeny of aldehyde dehydrogenase isozymes in the mouse: Evidence for a locus controlling the inducibility of the liver microsomal isozyme

Variation in the inducibility of the liver microsomal isozyme of aldehyde dehydrogenase (designated AHD-Cy) by phenobarbital administration was observed among inbred strains and linkage testing stocks of Mus musculus. The phenotypes were inherited in a normal Mendelian fashion with two alleles showing codominance at a proposed regulatory locus (designated Ahd-3r). Strain variation was also observed for the induction of liver AHD-Cy by 17-β-oestradiol administration to ovarectimized female mice. Moreover, this enzyme was elevated in activity by the administration of high (nonphysiological) levels of progesterone. Development studies showed that the liver and kidney AHD-Cy isozyme exhibited low activities in late-stage fetal and neonatal mice and reached adult levels by approximately 6 weeks of age.     

Genetics and ontogeny of aldehyde dehydrogenase isozymes in the mouse: Localization of Ahd-1 encoding the mitochondrial isozyme on chromosome 4

Electrophoretic variants for the mitochondrial isozyme of aldehyde dehydrogenase (AHD) have been observed in inbred strains and in Harwell linkage testing stocks of Mus musculus. F₁ (LVC×C57BL/Go) mice showed a codominant allele three-banded phenotype, which suggests a dimeric subunit structure (designated AHD-A₂). The anodal-migrating supernatant isozyme of AHD was electrophoretically invariant among the 23 inbred strains and stocks examined. The genetic locus encoding AHD-A₂ (suggested name Ahd-1) is localized on chromosome 4 and was mapped close to je (jerker) and Gpd-1 (encoding the liver and kidney isozyme of glucose-6-phosphate dehydrogenase). Ontogenetic analyses demonstrated that both AHD isozymes exhibited low activity in late fetal and early neonatal liver and kidney extracts, and reached adult levels within 3 weeks of birth.     

Genetic Regulation of Plasma Corticosterone Concentration and its Response to Castration and Allogeneic Tumour Growth in the Mouse

EXCESS fat deposition after castration is thought to be a response to hyperinsulinism induced by an increased level of adrenal glucocorticoids¹. Two mutant alleles, lethal yellow (A^y) and viable yellow (A^vy), at the agouti locus in the mouse induce excess fat deposition; the A^y allele has also been found to induce insulin resistance². Katsh³ concluded that in adrenalectomized KL strain mice a considerable portion of the high insulin tolerance depended on normal adrenal function. In the inbred YS/Wf and VY/Wf strains, both yellow pheno-types modulate serum insulin concentration⁴. Castration of inbred YS strain males causes a large decrease in serum insulin⁴, suggesting a possible relationship¹ to the concentration of adrenal glucocorticoids. Growth of allogeneic tumour cells has different effects on the serum insulin concentrations of YS and VY strain mice⁴.     

Isoenzymes of transaldolase in strains of Candida utilis and effect of different growth conditions

The isozymes of transaldolase have been investigated in six strains of Candida utilis. Four of these strains were cloned and in three of them more than one isozyme was found, indicating that mutltiple forms of the activity are present in the same cell. Cloned cells of one strain (C0U) were grown on three different media (YED, 1% dextrose, 1% yeast extract; YEX, 1% xylose, 1% yeast extract; AM, 1% dextrose, 1% ammonium sulfate, 0.025% yeast extract plus mineral salts) and analyzed for transaldolase isozyme patterns. Without autolysis the homogenates showed identical profiles with a single peak of activity characteristic of isozyme I. In spite of significantly different levels of pentose-phosphate pathway metabolites expected these patterns were not appreciably altered. Our data reveal a characteristic pattern for each strain and do not indicate a ready alteration of transaldolase isozymes as a response to changes in nutritional conditions.     

Analysis of neuraminidase isozyme phenotypes in mammalian tissues: an electrophoretic approach

A simple cellulose acetate electrophoretic method for visualizing mammalian neuraminidase isozymes has been developed. Application of the method with rat and mouse liver extracts reveals the presence of two distinct isozymes in each species. Each isozyme exhibits tremendous variation in activity between inbred strains. The two isozymes vary independently of one another suggesting that their activities are controlled by different genes. The neuraminidase phenotypes detected in these inbred strains via electrophoresis are consistent with published accounts of neuraminidase phenotypes determined fluorometrically in whole liver homogenates, but also indicate the presence of a second isozyme not perceived by this other procedure.     

The Ah Phenotype. Survey of Forty-Eight Rat Strains and Twenty Inbred Mouse Strains

Forty-four inbred and four randombred rat strains and 20 inbred mouse strains were examined for their Ah phenotype by determining the induction of liver microsomal aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity (EC 1.14.14.1) by intraperitoneal treatment with either β-naphthoflavone or 3-methylcholanthrene. All 48 rat strains were found to be Ah-responsive. The maximally induced hydroxylase specific activities of the ALB/Pit, MNR/Pit, MR/Pit, SHR/Pit, and Sprague-Dawley strains were of the same order of magnitude as the basal hydroxylase specific activities of the ACI/Pit, F344/Pit, OKA/Pit, and MNR/N strains. Six of the 20 mouse strains were Ah-nonresponsive (i.e. lacking the normal induction response and presumably lacking detectable amounts of the Ah receptor). The basal hydroxylase specific activities of the BDL/N, NFS/N, STAR/N, and ST/JN mouse strains were more than twice as high as the maximally induced hydroxylase specific activity of the CBA/HT strain.——To date, 24 Ah-nonresponsive mouse strains have been identified, out of a total of 68 known to have been characterized. The reasons for not finding a single Ah-nonresponsive inbred rat strain—as compared with about one Ah-nonresponsive inbred mouse strain found for every three examined—remain unknown.     

Genetic Organization of the agouti Region of the Mouse

The agouti locus on mouse chromosome 2 acts via the hair follicle to control the melanic type and distribution of hair pigments. The diverse phenotypes associated with various agouti mutations have led to speculation about the organization of the agouti locus. Earlier studies indicated that two presumed agouti alleles, lethal yellow (Ay) and lethal light-bellied nonagouti (ax), are pseudoallelic. We present genetic data showing probable recombination between Ay and three agouti mutations (at, a, and ax), which suggest that Ay is a pseudoallele of the agouti locus. The close linkage of an endogenous ecotropic murine leukemia provirus, Emv-15, to Ay provides a molecular access to genes at or near the agouti locus. However, previous studies suggested that the Emv-15 locus can recombine with some agouti alleles and therefore we analyzed mice from recombinant inbred strains and backcrosses to measure the genetic distance between various agouti alleles and the Emv-15 locus. Our data indicate that the Emv-15 locus is less than 0.3 cM from the agouti locus. These experiments provide a conceptual framework for initiating chromosome walking experiments designed to retrieve sequences from the agouti locus and give new insight into the genetic organization of the agouti region.     

Analysis of oocyte quality in recombinant inbred mouse strains developed from KE and CBA strains that differ in fertilization efficiency

Recombinant inbred (RI) mouse strains were developed from reciprocal crosses between two inbred strains differing in the proportion of fertilized ova (CBA, 100%; KE, 77%), to analyse the underlying factors. A correlation (r = 0.83, P < 0.01) between fertilization efficiency within 22 RI strains and after mating RI females with KE males proved that oocyte quality was involved. The following oocyte parameters were analysed in RI and progenitor strains: time of meiotic maturation, rapidity of enzymatic removal of egg investments, and proportion of fertilized ova with supplementary spermatozoa in the perivitelline space. Among the RI strains, high incidence of supplementary spermatozoa was correlated with lower efficiency of fertilization (r = -0.58, P < 0.05) and with slow meiotic maturation (r = -64, P < 0.01), suggesting that delayed maturation may affect oocyte ability of being fertilized by the first penetrating spermatozoon. However, significant correlations were also found between characters which coexist within the progenitor strains, but are not likely to be physiologically related; this suggests that RI strains have inherited large blocks of progenitor genomes, not disrupted by recombination. The strain distribution pattern (SDP) of the analysed traits revealed CBA-like, KE-like, and intermediate phenotypes, indicating that they are polygenically determined. No linkages were found between the studied traits and 12 enzymatic markers. However, the SDP for fertilization efficiency showed a preponderance of non-matching strains (15/19) in relation to agouti locus; the known instability of this chromosome region makes it possible that a putative linkage was disrupted by recombination when RI strains were created.     

Isozymes of NADP-dependent isocitrate dehydrogenase in normal and tumor tissues of various mouse strains and their hybrids

Normal tissues of DBA, CBA, CC57W, C3H, Balb/c, SHR mice and F1 hybrids CC57W/DBA appeared to differ in the ratios of mitochondrial and supernatant NADP-dependent isocitrate dehydrogenase (IDH). Tested inbred mice strains CC57W, C3H, SHR, Balb/c contain allelic form Idh-1a of supernatant IDH gene Idh-1, whereas allelic form Idh-1b is characteristic of mice strains DBA and CBA. In tumors IDH isozymes have the same mobility as do isozymes of homologous normal tissues; but their activity is lower. A high variability of each isozyme activity in the isozyme spectrum is revealed in various tissues of F1 hybrids CC57W/DBA. Allelic forms of gene Idh-1 were used as markers of normal and tumor cells for the experimental model: transplantation of sarcoma 37 (Idh-1a/Idh-1a) to subcutaneous tissue of the mouse strain DBA (Idh-1b/Idh-1b). It enables us to reveal isozymes of stromal cell in tumor IDH isozyme spectrum. The results indicate that the relation of normal and tumor isozymes vary in different tumors.     

Glutathione S-transferase isozymes in Aedes aegypti: Purification,characterization, and isozyme-specific regulation

Glutathione S-transferase (GST) isozymes were purified from the GG strain of Aedes aegypti, a strain having ≥4-fold higher total GST activity compared to the wild-type lab strain. Purification involved S-hexyl-glutathione affinity chromatography in high salt buffer, and GST specific elution with S-(p-bromobenzyl)-glutathione. Final purification was accomplished on DEAE-Sepharose. Two isozymes, GST-1b and GST-2 were purified using this procedure, and an additional isozyme, GST-1a, was partially purified. The GST-2 isozyme has one of the highest specific activities reported for a GST, with a specific activity of 739 μmol/min/mg using 1-chloro-2,4-dinitrobenzene (CDNB), and 16.4 μmol/min/mg using 3,4-dichloronitrobenzene (DCNB) as substrates. GST-2, GST-1a, and GST-1b were analyzed for amino acid composition and subjected to N-terminal sequencing. All three GSTs showed amino acid differences, especially among the nonpolar and polar amino acids. The amino acid composition of GST-1b was found to be more similar to GST 1-1 from Drosophila melanogaster than to GST-2 or GST-1a from Aedes aegypti. Only GST-2 gave N-terminal sequence data, raising the possibility that GST-1a and 1b are N-terminally blocked. The A. aegypti GST-2 showed amino acid sequence identity or similarity in all but one residue between residue numbers 31 through 41 compared to the D. melanogaster and Musca domestica GST 1-1 isozymes. The pattern of GST isozyme expression was analyzed in various tissues and stages of development of the GG and wild type strains using isozyme-specific antisera and substrates. GST-1a was constitutively overexpressed in all tissues examined in the GG strain compared to the wild type strain. The expression of GST-1b was similar in both strains for all tissues and developmental stages examined. GST-2 was constitutively overexpressed in head, thorax and abdomen, but was not detected in ovaries of the GG strain. These results suggest that elevated GST activity in the GG strain is due to constitutive overexpression of GST-2 and GST-1a. GST-1a, GST-1b and GST-2 apparently are the products of 3 independently regulated genes and appear to be expressed in a tissue-specific manner.     

Lactate dehydrogenase isozymes in xenotropic virus producing mice

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) isozymes were compared in three inbred strains of mice, and two strains of wild mice, as well as the F1 hybrids and other genetic crosses involving two of the inbred strains. The strains examined were NZB/B1NJ, 129/J and C57BL/6J, Mus musculus molossinus and M. musculus castaneus. Genetic crosses were made between the xenotropic virus-producing NZB and the non-virus producing 129/J mice. Tissue specificity of LDH in these strains was studied using homogenates of kidney, liver, spleen and thymus. Polymorphism of the enzyme was studied by agarose gel electrophoresis. Enzyme polymorphism in the tissues of NZB and 129/J has not been previously reported. The liver and spleen tissues of 129/J showed the absence of LDH-1 and LDH-2 isozymes. Thymic homogenates of NZB showed a lack of expression of LDH-1, LDH-2 and LDH-3 isozymes. The F1, F2 and the backcross progeny from genetic crosses involving NZB, and 129/J mice showed an isozyme pattern more similar to the non-virus-producing 129/J strain than the virus-producing NZB. Evidence of genetic regulation at the LDH-B subunit appears to be the reason for the differential expression of the isozymes in NZB and 129/J strains. The other inbred strain of mice, C57BL/6J, also showed a greater similarity to the 129/J strain than NZB. The two strains of wild mice were similar in their expression of LDH-isozymes between each other and to the 129/J strain, with respect to the liver and spleen tissues.     

Genetic Linkage between the AH Locus and a Major Gene That Inhibits Susceptibility to Audiogenic Seizures in Mice

Mice of the DBA/2 (D2) strain are highly susceptible to sound-induced seizures at 21 days of age; whereas, mice of the C57BL/6 (B6) strain are resistant to these seizures. Although the difference in susceptibility to audiogenic seizures (ASs) between these two strains is inherited as a multiple-factor trait, an association was observed between susceptibility to ASs and the Ah locus. The Ah locus controls the inducibility of aryl hydrocarbon hydroxylase (AHH) activity by a number of aromatic hydrocarbons. B6 mice carry the Ah^b allele and have inducible AHH activity; whereas, D2 mice carry the Ah^d allele and have noninducible activity. Inducibility is inherited as a Mendelian dominant trait in crosses between these strains. Mice carrying the Ah^b allele are generally less susceptible to ASs at 21 days of age than are mice carrying the Ah^d allele. The combined results from B6 x D2 recombinant inbred strains, congenic strains (where the Ah^b allele was placed into the D2 genome and the Ah^d allele placed into the B6 genome), the B6D2F₁ x D2 backcross generation, and a random survey of various inbred strains, suggest that the association between these two traits is due to genetic linkage, rather than to pleiotrophy or to chance. A major gene that inhibits susceptibility to ASs appears to be closely linked to the Ah locus. This gene has been designated Ias, for inhibition of ASs. A large portion of the genetic variability of AS susceptibility may be due to the segregation of Ias.     

Kraft Pulp Bleaching and Delignification by Dikaryons and Monokaryons of Trametes versicolor

The ability of 10 dikaryotic and 20 monokaryotic strains of Trametes (Coriolus) versicolor to bleach and delignify hardwood and softwood kraft pulps was assessed. A dikaryon (52P) and two of its mating-compatible monokaryons (52J and 52D) derived via protoplasting were compared. All three regularly bleached hardwood kraft pulp more than 20 brightness points (International Standards Organization) in 5 days and softwood kraft pulp the same amount in 12 days. Delignification (kappa number reduction) by the dikaryon and the monokaryons was similar, but the growth of the monokaryons was slower. Insoluble dark pigments were commonly found in the mycelium, medium, and pulp of the dikaryon only. Laccase and manganese peroxidase (MnP) but not lignin peroxidase activities were secreted during bleaching by all three strains. Their laccase and MnP isozyme patterns were compared on native gels. No segregation of isozyme bands between the monokaryons was found. Hardwood kraft pulp appeared to adsorb several laccase isozyme bands. One MnP isozyme (pI, 3.2) was secreted in the presence of pulp by all three strains, but a second (pI, 4.9) was produced only by 52P. A lower level of soluble MnP activity in one monokaryon (52D) was associated with reduced bleaching ability and a lower level of methanol production. Since monokaryon 52J bleached pulp better than its parent dikaryon 52P, especially per unit of biomass, this genetically simpler monokaryon will be the preferred subject for further genetic manipulation and improvement of fungal pulp biological bleaching.     

Inheritance of Adrenal Phenylethanolamine N-Methyltransferase Activity in the Rat

Phenylethanolamine N-methyltransferase (PNMT) is the enzyme that catalyzes the S-adenosyl-L-methionine-dependent methylation of (-)norepinephrine to (-)epinephrine in the adrenal medulla. Adrenal PNMT activity is markedly different in two highly inbred rat strains; enzyme activity in the F344 strain is more than fivefold greater than that in the Buf strain. Initial characterization of the enzyme in the two inbred strains reveals evidence for catalytic and structural differences, as reflected in dissimilar Km values for the cosubstrate (S-adenosyl-L-methionine) and prominent differences in thermal inactivation curves. To assess adrenal PNMT activity in an F344 X Buf pedigree, we employed a statistical procedure to test for one- and two-locus hypotheses in the presence of within-class correlations due to cage or litter effects. The PNMT data in the pedigree are best accounted for by segregation at a simple major locus superimposed upon a polygenic background; data obtained from the biochemical studies suggest that the major locus is a structural gene locus.     

Origin of cytokinin-and auxin-autonomy and changes in specific proteins in tobacco callus tissue

On the basis of earlier data it was suggested that the induction of cytokinin autonomy might be accompanied by disorders in plastid function and a decrease in cytokinin utilization. In the work presented below the formation of chlorophyll and the isozyme patterns of nine enzymes, some of which are known to be localized in plastids, were compared in tobacco callus tissues differing in their hormonal requirements. Tissues either not requiring cytokinin or both auxin and cytokinin for their growth, contained a lower amount of chlorophyll than the cytokinin-and auxin-dependent strain. The number of isozymes of glucose-6-phosphate and NADP-malate dehydrogenase (i.e. enzymes which are known to be located in plastids) was reduced from four in the cytokinin-and auxin-dependent strain to two and one in the two cytokinin-autonomous strains, respectively. The fully habituated tissue contained an additional isozyme of NADP-malate dehydrogenase. The total number of isozymes of the remaining enzymes (NAD-malate dehydrogenase, peroxidase, esterase and a-and β-galactosidase) either was decreased or not changed in the cytokinin autonomous strains. The exception was an additional anodic peroxidase in one strain. The number of these isozymes in tissue habituated with respect to both auxin and cytokinin either remained the same or increased. Tobacco callus strains with altered requirements for growth regulators contained some new isozymes which were not present in any other strain and some isozymes present in other strains were absent. These differences are discussed in relation to the possible role of plastid function disorder associated with habituation.