A structural gene (Hdc-s) for mouse kidney histidine decarboxylase

The concentration of mouse kidney histidine decarboxylase (HDC) is modulated by estrogen, testosterone, and thyroxine in a tissue-specific manner. Variation in HDC levels between strains of mice can be used to investigate the genetic regulation of (i) enzyme structure, (ii) tissue specific expression, and (iii) induction and repression by hormones. Variation in the structure of HDC between different inbred strains of mice affecting its K _m for the cofactor pyridoxal-5-phosphate (PLP) and its heat stability has been discovered. The alternative phenotypes are additively inherited in crosses and the heat stability difference is due to alleles of a single structural gene, Hdc-s, which segregate among the BXD and BXH recombinant inbred strains. The allele Hdc-s ^b determines the heat-stable phenotype (C57BL substrains), and the allele Hdc-s ^d the heat-labile phenotype (DBA/2 and C3H/He strains). The alleles of the structural gene cosegregate with alleles of a regulatory gene previously named Hdc (determining kidney enzyme concentration); there were no recombinants among 38 RI strains. Therefore the two loci are less than 0.685 cM apart and comprise part of the HDC gene complex, [Hdc], on chromosome 2 of the mouse.This work was supported in part by an SERC studentship to S.A.M. and an MRC project grant to G.B.     

Histidine decarboxylase phenotypes of inbred mouse strains: A regulatory locus (Hdc) determines kidney enzyme concentration

Mouse kidney histidine decarboxylase (HDC) provides a model system to study genetic control of a hormone-regulated enzyme (inducible by estrogen and thyroxine; repressible by testosterone). Five major HDC phenotypes scored on the basis of (i) enzyme activity and (ii) the difference in activity between the sexes (females usually higher than males) have been discovered by screening 38 strains of mice. One genetic difference between high-activity strains (DBA/2 and C3H/He) and low-activity strains (C57BL/6 and C57BL/10) has been examined in detail. The phenotypic difference segregates as a single gene in both conventional crosses and between recombinant inbred (RI) strains. Immunoprecipitation has shown that the activity difference is due to an alteration in the number of enzyme molecules. The phenotypic difference between high and low strains can therefore be attributed to different alleles of a single regulatory locus, Hdc; the alleleHdc ^d determines low HDC concentration, and the allele Hdc ^d high concentration. Hdc has been mapped to chromosome 2 using data from both comparisons of strain distribution patterns of previously mapped loci within RI strains and a conventional three-point cross. The probable gene order is B2m-pa-Hdc, with map distances of 3.1±1.7 and 2.0±1.4 cM, respectively.This work was supported by an MRC project grant to Grahame Bulfield, an SERC research studentship to S. A. M. Martin, and NIH Research Grant GM 18684 from the National Institute of General Medical Sciences to B. A. Taylor. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory animal care.     

Dominance relationships among mutant alleles of regulatory gene araC in the Escherichia coli B/R L-arabinose operon.

The araBAD operon of Escherichia coli B/r is positively and negatively regulated by the araC+ regulatory protein. Mutations in gene araC can result in a variety of different regulatory phenotypes: araC null mutants (those carrying a null allele exhibiting no repressor or activator activity) are unable to achieve operon induction; araC-constitutive (araCc) mutants are partially constitutive, inducible by D-fucose, and resistant to catabolite repression; araCh mutants are hypersensitive to catabolite repression; and araCi mutants are resistant to catabolite repression. Various mutant alleles of gene araC were cloned into a derivative of plasmid pBR322 by in vivo recombination. Various heterozygous araC allelic combinations were constructed by transformation. Analysis of isomerase (araA) specific activity levels under various growth conditions indicated the following dominance relationships with regard to sensitivity to catabolite repression: araCh greater than araC+ greater than (araCc and araCi) greater than araC. It was concluded that the araCh protein may form a repressor complex that is refractory to removal by cyclic AMP receptor protein-cyclic AMP complex. This was interpreted in terms of the known nucleoprotein interactions between ara regulatory proteins and ara regulatory DNA.     

Nitrogen regulation of amino acid utilization by Neurospora crassa.

The production of an extracellular deaminase activity involved with the utilization of amino acids as sole sources of nitrogen is under the control of the nit-2 locus of Neurospora crassa. This locus is the sole major nitrogen regulatory locus described for N. crassa and is believed to encode a positive effector required for induction of activities involved with the utilization of alternate nitrogen sources. Production of deaminase activity requires the lifting of nitrogen metabolite repression, the presence of a functional nit-2 gene product, and specific induction by amino acids. Additional parameters of enzyme production are described.     

Gene dosage effects on the synthesis of maltase in yeast.

Inbred strains of Saccharomyces cerevisiae carrying MAL1, MAL2, or MAL6 in a common background were used to construct (i) homo- or heterozygous diploids carrying one or two active alleles of a single MAL locus (MAL1, MAL2, or MAL6) and (ii) triploids carrying one, two, or three active alleles of MAL2. The diploid and triploid strains were used to investigate gene dosage effects of the differential rate of maltase synthesis (delta enzyme activity/delta growth) and the kinetics of induction (for MAL2). All three MAL loci exhibited a gene dosage effect on the differential rate of maltase synthesis; MAL2 also exhibited a gene dosage effect on the kinetics of induction. The dosage effects of the MAL1 and MAL6 loci were additive, but the effects of the MAL2 locus were not; the magnitude of the MAL2 gene dosage effect decreased with increasing dosage. These results are compatible with the current genetic evidence that the MAL genes are regulatory loci if the product(s) of the MAL1 and MAL6 locus is produced in limiting amounts but the product(s) of the MAL2 locus is produced in excess, except at very low genes dosages.     

Polymorphism of cattle prolactin gene: microsatellites, PSR-RFLP]

In the samples of Russian Ayrshire and Gorbatov Red cattle breeds, distribution of frequencies of prolactin (PRL) gene alleles generated due to the presence of polymorphic RsaI site in exon 3 were studied. In the breeds, the frequencies of the B allele of the PRL gene (with RsaI(+) site) detected by the PCR-RFLP method were 14.1 and 8.6%, respectively. In Black Pied, Ayrshire and Gorbatov Red cattle breeds, variation of the microsatellite dinucleotide repeat in the regulatory region of the gene PRL was also studied. Gorbatov Red breed was monomorphic at the microsatellite locus with the only allele 164 bp in length. Two alleles (164 bp and 162 bp) were detected in the other breeds studied. The frequencies of 164-bp allele of the microsatellite locus were 93.7 and 90.0% in Black Pied and Ayrshire breeds, respectively. In Gorbatov Red breed of dairy type with good beef qualities and low milk-fat yield, lower level of heterozygosity for PRL gene was demonstrated compared to Ayrshire and Black Pied breeds with high milk-fat yield. In three cattle breeds, higher mean estimate of polymorphism information content of PCR-RFLP in exon 3 (PIC = 0.21) was revealed compared with the same estimate (PIC = 0.09) for the microsatellite locus variability in the regulatory region of the PRL gene. Characteristics of allele B distribution of the PRL gene in the representatives of the Bovidae family are considered.     

Plasma paraoxonase polymorphism: a new enzyme assay, population, family, biochemical, and linkage studies.

Plasma paraoxonase hydrolyzes paraoxon, the principal metabolite of the insecticide parathione. A genetic polymorphism for enzyme activity has been previously demonstrated. We describe a new assay based on the differential inhibition by EDTA of plasma paraoxonase from persons with the high-activity allele (PX*H) that suggests a trimodality of activity levels in population studies. The gene frequency of the low activity allele (PX*L) in 531 Seattle blood donors of European origin was .7207. Family studies were consistent with codominant autosomal inheritance of two alleles, PX*L (low) and PX*H (high), coding for products with different activity levels. Biochemical measurements of sera from presumed homozygotes for the two different alleles revealed minor physicochemical differences suggestive of a structural difference between the allelic products. No evidence for linkage of the paraoxonase locus with any of 19 polymorphic markers would be detected.     

Gene activation of alcohol dehydrogenase in danio hybrids

The regulation of alleles encoding the enzyme alcohol dehydrogenase (ADH) was investigated in F1Brachydanio hybrids (zebra danio female x spotted danio male) by acrylamide gel electrophoresis. Both parental species showed a single, cathodal band of species-specific ADH. During development at 26 degrees C, hybrid fry showed a preferential activation of the maternally derived Adh allele. It is suggested that the low activity of the paternally derived allele may result from an incompatibility between maternal regulatory factors and the paternal regulative element controlling gene expression.     

Zn++ stimulation of the induction of alcohol dehydrogenase by anaerobiosis in roots of Zea mays L.

Summary Zn⁺⁺ at an optimum concentration of 5×10^–4 M caused a two fold stimulation in the level of alcohol dehydrogenase (ADH) induced by anaerobic conditions. Isozymes specified by different genes and alleles show disproportionate increases in activity, such that, unequal representation of gene products while not eliminated, is invariably reduced by Zn⁺⁺ treatment. Thus in the case of alleles at the Adh-1 locus, there was a greater increase in the protein subunit specified by the Adh-1S allele. From previous work (Fischer and Schwartz, 1973) this protein is known to have a reduced affinity for Zn⁺⁺. This suggests that zinc availability during ADH induction is limiting and may provide an alternative to the cis-linked regulatory gene model proposed by Schwartz (1971) to explain the unequal expression of genes and alleles.     

Reversion at the HIS1 Locus of Yeast

The his1 gene (chromosome V) of Saccharomyces cerevisiae specifies phosphoribosyl transferase (E.C.2.4.2.17), the first enzyme of histidine biosynthesis. This hexameric enzyme has both catalytic and regulatory functions. The spontaneous reversion rates of seven his1 mutations were studied. The reversion rates of the alleles at the proximal end of the locus (relative to the centromere) were about 50-fold higher than distal alleles. Spontaneous reversion to prototrophy was studied in diploids homoallelic for each of the seven his1 mutations. Based on tetrad analysis, the prototrophy revertants could be assigned to three classes: (1) revertant tetrads that carried a prototrophic allele indistinguishable from wild type; (2) revertant tetrads that carried a prototrophic allele characterized by histidine excretion and feedback resistance; and (3) revertant tetrads that did not contain a prototrophic spore, but rather a newly derived allele that complemented the original allele intragenically. Four of the seven his1 mutations produced the excretor revertant class, and two mutations produced the complementer revertant class. The significance of these findings to our understanding of gene organization and the catalytic and regulatory functions of gene products are discussed.     

Mapping, complementation, and targets of the cysteine protease actinidin in kiwifruit

Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin.     

Genetic control of glucuronidase induction in mice

The β-glucuronidase activity of mouse kidney proximal tubule cells increases rapidly after administration of dihydrotestosterone. Several inbred mouse strains show an approximately fourfold greater response in enzyme activity than the majority of strains, although both groups have similar uninduced kidney glucuronidase activity. This difference is maintained throughout a three-week induction period. It is not accounted for by a difference in any of the physiological parameters (e.g. hypertrophy, inducer specificity, enzyme secretion) associated with enzyme induction. The difference is specific to glucuronidase; assays of other androgen-indueible enzymes showed no difference between the two groups. Induction does not involve a change in enzyme structure since basal and induced glucuronidase have identical thermal stability and immunochemical reactivity.Rates of enzyme synthesis were determined by assaying the incorporation of radiolabeled leucine into antibody-purified glucuronidase. The rate of enzyme synthesis increases after induction, and the more rapidly inducing strains have a correspondingly greater increase in the rate of enzyme synthesis.The inducibility difference between the two classes of inbred strains segregates as a single Mendelian trait in both backcross and F₂ progeny. Recombination studies with a coat color mutation closely linked to the enzyme structural gene and with a mutant of the enzyme structural gene altered in electrophoretic mobility showed that the inducibility gene, called Gur, maps in the region of the glucuronidase structural gene. Tests in heterozygotes showed that the Gur locus acts cis, affecting only the rate of synthesis of glucuronidase coded by the structural gene residing on the same chromosome.     

Genetic control of heat sensitivity and activity level of murine arylsulfatase B

BALB/c male mice possess twofold higher kidney p-nitrocatechol-SO4 arylsulfatase B than do A/HeJ male mice; however, their liver arylsulfatase activities are comparable. Twentyfold-purified kidney arylsulfatases B from these two strains have similar Michaelis constants, electrophoretic mobilities, pH optima, and inhibitor profiles; however, the BALB/c enzyme is more heat stable than the A/HeJ enzyme. BALB/c, C3H/HeJ, DBA/2J, and SWR/J mice share an autosomal allele, As-1a, which apparently determines the heat-stable arylsulfatase B, while A/HeJ and C57BL/6J mice possess the As-1b allele, which determines the heat-sensitive enzyme. A second autosomal locus, Asr-1, determines liver arylsulfatase B activity. C57BL/6J mice carry the Asr-1a allele, which results in high liver activities, while C3H/HeJ mice are homozygous for the low-activity allele, Asr-1b. Male mice generally have 30-40% higher kidney activities than females; however, female kidney arylsulfatase activities rise and actually surpass those of males during late pregnancy and lactation.     

Salinity related physiological and genetic differences between populations of Mytilus edulis

Aminopeptidase-I, the enzymatic product of the Lap locus, liberates N-terminal neutral and aromatic amino acids from oligopeptides. The enzyme is associated with the brush border of the intestine and the extensive lysosomal system in the digestive tubule cells; the enzyme functions in oligopeptide degradation and possibly amino acid transport. The Lap locus is genetically polymorphic and allele frequencies differ between populations according to environmental salinities.
Using cell-free lysosomes, we show salinity related differences in both lysosomal membrane latency and lysosomal and cytosolic free-amino acid concentrations. Differences in the response of the lysosomal aminopeptidase-I enzyme to differences or changes in salinity are discussed.
Antibodies against aminopeptidase-I enzyme were used to demonstrate that alleles of the Lap locus exhibit different specific activities per unit enzyme concentration. In oceanic populations, the concentrations of aminopeptidase-I enzyme of individual phenorypes are equal, suggesting that variation of specific activity among phenorypes is due to allele specific differences in catalytic efficiency. In estuarine populations, total enzyme activity is lower, which is partially due to a significantly lower concentration of aminopeptidase-I enzyme of one homozygote genotype.
The consequences of a change in environmental salinity can be measured on the biochemical, physiological, and population genetic levels. We discuss the possible mechanisms by which salinity variations are responsible for the genetic polymorphism of aminopeptidase-I.     

Isolation and characterization of mutants that produce the allantoin-degrading enzymes constitutively in Saccharomyces cerevisiae.

Degradation of allantoin, allantoate, or urea by Saccharomyces cerevisiae requires the participation of four enzymes and four transport systems. Production of the four enzymes and one of the active transport systems is inducible; allophanate, the last intermediate of the pathway, functions as the inducer. The involvement of allophanate in the expression of five distinct genes suggested that they might be regulated by a common element. This suggestion is now supported by the isolation of a new class of mutants (dal80). Strains possessing lesions in the DAL80 locus produce the five inducible activities at high, constitutive levels. Comparable constitutive levels of activity were also observed in doubly mutant strains (durl dal80) which are unable to synthesize allophanate. This, with the observation that arginase activity remained at its uninduced, basal level in strains mutated at the DAL80 locus, eliminates internal induction as the basis for constitutive enzyme synthesis. Mutations in dal80 are recessive to wild-type alleles. The DAL80 locus has been located and is not linked to any of the structural genes of the allantoin pathway. Synthesis of the five enzymes produced constitutively in dal80-1-containing mutants remains normally sensitive to nitrogen repression even though the dal80-1 mutation is present. From these observations we conclude that production of the allantoin-degrading enzymes is regulated by the DAL80 gene product and that induction and repression of enzyme synthesis can be cleanly separated mutationally.     

The GLN1 Locus of SACCHAROMYCES CEREVISIAE Encodes Glutamine Synthetase

Among 41 yeast glutamine auxotrophs, complementation analysis defined a single gene, GLN1, on chromosome 16 between MAK3 and MAK6. Half of the alleles fell into two intragenic complementation classes. No clustering of complementing alleles was found in a fine structure map. Altered glutamine synthetase subunits, including nonsense fragments and charge variants, were identified in several of the mutants, indicating that GLN1 is the structural gene for this enzyme. Negative complementation was observed for almost every allele associated with a protein product and all gln1/+ heterozygotes displayed reduced susceptibility to ammonia repression of the remaining glutamine synthetase activity. This latter observation is explained by the hypothesis that ammonia represses the enzyme only through its metabolism to glutamine. A basis for the two gln1 complementation classes is proposed.