Enzyme activity and distribution of adenosine deaminase types in a population of central Italy

Blood samples from 718 unrelated individuals born in municipalities of the 'Alto Maceratese' region in central Italy were examined for adenosine deaminase (ADA). The allele frequencies for ADA*1 and ADA*2 were 92.48 and 7.45%, respectively. One case of the rare ADA 5-1 phenotype was observed. The observed phenotype distribution agreed well with the one expected assuming a Hardy-Weinberg equilibrium. Enzyme activity was measured in red blood cells from individuals with different phenotypes. The relationships between the enzyme activity of the phenotypes was found to be ADA 1 greater than ADA 2-1 greater than ADA 2 greater than ADA 5-1.     

Characterization of ACP1TIC-1, an electrophoretic variant of erythrocyte acid phosphatase restricted to the Ticuna Indians of central Amazonas.

A new variant of erythrocyte acid phosphatase, designated ACP1TIC-1, is characterized by a more cathodal electrophoretic mobility than any of the common polymorphic phenotypes, both in the presence and absence of tricarboxylic acids. Individuals of the ACP1TIC-1 phenotype have a level of enzyme activity (4.8 +/- 0.1 mumol/g hemoglobin per min) similar to individuals of the ACP1A phenotype, although no differences in Km values were observed or is the extent of phosphate inhibition different between the ACP1TIC-1 and the ACP1B variants. The thermostability of the enzyme is less than that observed for any of the common variants. The TIC-1 variant is activated by adenine and inhibited by folic acid to the same extent as the type-A enzyme, while the stimulation of the activity of the TIC-1 enzyme by hypoxanthine and the inhibition of it by uric acid is similar to that for the B enzyme. Thus, the TIC-1 variant has a unique combination of kinetic properties, seeming to be a hybrid of A-type and B-type characteristics.     

Population genetic studies of the Philippine Negritos. I. A pilot survey of red cell enzyme and serum protein groups

Electrophoretic surveys of red cell enzyme and serum protein systems representing 21 genetic loci were carried out on 129 blood samples of the Negritos of Pampanga, Central Luzon, the Philippines. Nine (out of 16) red cell enzyme loci and four (out of five) serum protein loci showed polymorphic variation. Low frequencies of ACP 1A, GPTs1, ESD2, and Hp1, and a markedly high frequency of PGM12 were contrasted to those in non-Negrito Filipinos. Variant ESD phenotypes with a slowly migrating isozyme occurred in high frequency. The new allele designated as ESD3Negrito (ESD3N) had a frequency of .10 +/- .019. In AK, a variant phenotype indistinguishable from AK 2-1 was observed in 14% of the sample. In the Gc system, a fast migrating variant was discovered in high frequency which was distinct from Gc Ab and Gc J. The variant allele, denoted GcNegrito (GcN), had a frequency of .21 +/- .025. A relatively high degree of allelic diversity in the Negrito sample was also suggested by the average heterozygosity for 21 loci screened (.165), which is compared to that of the Japanese population (.140).     

Tissue-Specific and Complex Complementation Patterns in the Punch Locus of DROSOPHILA MELANOGASTER

Mutations in the Punch locus result in loss of GTP cyclohydrolase activity, but all mutations do not affect the enzyme in the same way. There are at least three classes of Punch mutations. One class results in a dominant eye color, recessive lethal phenotype. A second class of mutations also causes a recessive lethal phenotype, but heterozygous mutants have normal eye color. They show loss of GTP cyclohydrolase function in all tissues where activity can be measured. Alleles comprising a third class are recessive eye color mutations that are homozygous viable. Individuals with this third type of mutation show loss of enzyme activity in the eye, but show normal or near-normal activity elsewhere. In order to examine the organization and function of this locus further, we have performed interallelic complementation tests on 25 Punch mutations, monitoring viability and enzyme activity in prepupae and adults. Most allele combinations are lethal. Those that complement do so in ways that are tissue-or stage-specific and unpredictable. Tests of mutants with tissue-specific phenotypes and of individuals mutant for complementing Punch lethal alleles lead us to conclude that Punch is a complex locus, both with respect to its organization and to its products.     

Molecular analysis of esterase D polymorphism

We have analyzed the esterase D (EsD) polymorphism at the nucleic acid level. Two common alleles, EsD¹ and EsD², are characterized by the substitution of one amino acid (Gly-to-Glu), which is caused by the point mutation of one nucleotide (G-to-A). Individuals exhibiting the EsD1 and EsD 2 phenotypes are homozygotes for EsD 1 and EsD 2 cDNAs, respectively. Individuals showing the EsD 2-1 phenotype have two kinds of cDNAs, viz., EsD 1 and EsD 2. The point mutation difference between the cDNAs of the EsD¹ and EsD² alleles results in a different SspI digestion site. A restriction fragment length polymorphism caused by this difference with respect to the SspI digestion site makes it possible to determine the EsD phenotype using DNA samples extracted from forensic materials with no EsD enzymatic activity.     

The Magnitude and Specificity of Influenza A Virus-Specific Cytotoxic T-Lymphocyte Responses in Humans Is Related to HLA-A and -B Phenotype

The repertoire of human cytotoxic T-lymphocytes (CTL) in response to influenza A viruses has been shown to be directed towards multiple epitopes, with a dominant response to the HLA-A2-restricted M1(58-66) epitope. These studies, however, were performed with peripheral blood mononuclear cells (PBMC) of individuals selected randomly with respect to HLA phenotype or selected for the expression of one HLA allele without considering an influence of other HLA molecules. In addition, little information is available on the influence of HLA makeup on the overall CTL response against influenza viruses. Here, the influenza A virus-specific CTL response was investigated in groups of HLA-A and -B identical individuals. Between groups the individuals shared two or three of the four HLA-A and -B alleles. After in vitro stimulation of PBMC with influenza virus, the highest CTL activity was found in HLA-A2(+) donors. A similar pattern was observed for the precursor frequency of virus-specific CTL (CTLp) ex vivo, with a higher CTLp frequency in HLA-A2-positive donors than in HLA-A2-negative donors, which were unable to recognize the immunodominant M1(58-66) epitope. In addition, CTL activity and frequency of CTLp for the individual influenza virus epitopes were determined. The frequency of CTLp specific for the HLA-B8-restricted epitope NP(380-388) was threefold lower in HLA-B27-positive donors than in HLA-B27-negative donors. In addition, the frequency of CTLp specific for the HLA-A1-restricted epitope NP(44-52) was threefold higher in HLA-A1-, -A2-, -B8-, and -B35-positive donors than in other donors, which was confirmed by measuring the CTL activity in vitro. These findings indicate that the epitope specificity of the CTL response is related to the phenotype of the other HLA molecules. Furthermore, the magnitude of the influenza virus-specific CTL response seems dependent on the HLA-A and -B phenotypes.     

Genotype-phenotype correlations in Lesch-Nyhan disease: moving beyond the gene

Lesch-Nyhan disease and its attenuated variants are caused by mutations in the HPRT1 gene, which encodes the purine recycling enzyme hypoxanthine-guanine phosphoribosyltransferase. The mutations are heterogeneous, with more than 400 different mutations already documented. Prior efforts to correlate variations in the clinical phenotype with different mutations have suggested that milder phenotypes typically are associated with mutants that permit some residual enzyme function, whereas the most severe phenotype is associated with null mutants. However, multiple exceptions to this concept have been reported. In the current studies 44 HPRT1 mutations associated with a wide spectrum of clinical phenotypes were reconstructed by site-directed mutagenesis, the mutant enzymes were expressed in vitro and purified, and their kinetic properties were examined toward their substrates hypoxanthine, guanine, and phosphoribosylpyrophosphate. The results provide strong evidence for a correlation between disease severity and residual catalytic activity of the enzyme (k(cat)) toward each of its substrates as well as several mechanisms that result in exceptions to this correlation. There was no correlation between disease severity and the affinity of the enzyme for its substrates (K(m)). These studies provide a valuable model for understanding general principles of genotype-phenotype correlations in human disease, as the mechanisms involved are applicable to many other disorders.     

A new genetic variant of galactose-1-phosphate uridyl transferase

Summary The enzyme galactose-1-phosphate uridyl transferase (E.C. 2.7.7.12), which has an important function in the metabolism of galactose, exists in multiple molecular forms. The different phenotypes are genetically determined. They can be distinguished according to their electrophoretic mobility. The enzymatic activity of the different gene products varies within certain limits. A new phenotype of the enzyme has been detected in the red cells of a healthy individual. The electrophoretic migration of this phenotype is slower compared to the wild type and its enzymatic activity is lower, but still sufficient as not to cause galactosemia. An extensive family study revealed that the rare gene is inherited according to mendelian law. Independently the same gene product has been detected in two other, nonrelated individuals out of a total of 1668 samples tested. The gene frequency can therefore be estimated to 0.0009 in the Swiss population. We suggest that the new type be called Berne variant of galactose-1-phosphate uridyl transferase.     

Effects of deoxycoformycin in mice. II. Differences between the drug sensitivities and purine metabolizing enzymes of transplantable lymphomas of varying immunologic phenotypes

Transplantable BALB/c and AKR lymphomas of different cell surface immunologic phenotypes have distinctive patterns of response to the ADA inhibitor DCF in vivo and in vitro. BAL 9, a lymphoma of the Lyt-1+,2+ T cell phenotype, was the most sensitive to DCF in vivo, and its DNA synthesis was inhibited more than 95% when cultured in the presence of dAr and DCF in vitro. This was correlated with a 10-fold increase in dATP content. The ADA and AMPDA activities were both high. Two lymphomas of the Lyt-1-,2+ T cell phenotype, BAL 5 and AKTB - lt , as well as two B cell phenotype lymphomas, A20 .3 and AKTB -lb, were all moderately inhibited in their in vivo growth if enough DCF was administered. However, their DNA synthesis in vitro was only inhibited 8 to 24% by dAr and DCF, there was only a twofold increase in the accumulation of dATP, and ADA and AMPDA activities were both low in the two BALB/c lymphomas tested. BAL 13, the only lymphoma of the Lyt-1+,2- phenotype examined, was completely resistant to DCF in vivo and in vitro. When cultured in the presence of dAr and DCF there was a transient increase in dATP content, followed by an abrupt decline. AMPDA activity was five to seven times greater than in the other lymphomas tested. ADA activity was moderate. The activities of 5' nucleotidase and of adenosine kinase were low and approximately equal in all the BALB/c lymphomas. These results suggest that the response to DCF by lymphomas of various immunologic phenotypes can be correlated with their nucleoside metabolism. The sensitivity of BAL 9 and the resistance of BAL 13 to DCF are correlated with their tendency to accumulate dATP and with their AMPDA and ADA activity ratios. The moderate sensitivity to DCF in vivo of the other T and B cell lymphomas, however, could not be clearly explained by any of the in vitro parameters thus far investigated, and this suggests that mechanisms inhibiting lymphoma proliferation other than dATP accumulation may be operating.     

Molecular evidence that the esterase-D gene lies proximal to the retinoblastoma susceptibility locus in chromosome region 13q14

Summary Somatic cell hybrids have been created between transformed mouse 3T3 cells and fibroblasts from a retino-blatoma patient with normal red-cell esterase-D (ESD) levels and a constitutional deletion of chromosome region 13q14-q31. In one subclone, which has retained the deletion chromosome but not the homologous normal copy, we have demonstrated the presence of the human ESD gene sequence. The breakpoint in this patient therefore must have occurred between the ESD gene and the retinoblastoma (Rb) predisposition locus. We have also been able to demonstrate that the ESD gene lies proximally to be the Rb gene in region 13q14. The recently isolated 4.7R cDNA gene sequence was absent from the deletion-containing hybrid, a finding consistent with the hypothesis that this sequence represents the Rb gene itself.     

Gross Genetic Dissection and Interaction of the Chromosomal Region 95e;96f of Drosophila Melanogaster

Making use of deficiencies, inversions and translocations, we have genetically dissected the region 95E to 96F of Drosophila melanogaster. We localized cytologically the loci abnormal spindle (asp: 3-85.2: 96A20-25;96B1-10) and M(3)96C2 (96C1;96C5). We have also found several new phenotypes associated with lesions in the 95E to 97B region: (1) Minute(3)96A (M(3)96A) is a haplo-insufficient phenotype of thin and short bristles presented by individuals deficient for the region 95E6-8;96A1-5. (2) abdominal-one reduced (aor) shows two different phenotypes associated with the distal breakpoint of In(3R)Ubx7L (89E;96A1-7). One is the increase of the Ubx phenotype, but its effect requires the presence of lesions in Ubx. The other phenotype is a drastic reduction or disappearance of the first abdominal segment. Both phenotypes might be due to lesions in the same gene. (3) metaphase arrest (mar) is associated with the breakpoint of the T(Y;3)B197 (96B1-10) and produces a phenotype typical of mitotic mutants with arrest of the cell cycle during prometaphase or metaphase. There is another region localized in 97B which interacts with asp: in a background homozygous for asp, three doses of this region enhance the asp phenotype.     

Association of the CHE2 locus with body mass index and butyrylcholinesterase activity

The butyrylcholinesterase (BChE; EC 3.1.1.8) activities of two electrophoretic bands of the CHE2 C5+ phenotype--C5 and C(OF) (other forms)--were quantified by densitometry in 100 individuals. The activity data suggested that, in addition to determining C5, the CHE2*C5+ allele also increases the level of other BChE forms. Since the relative activity of C5 showed the highest correlation coefficient with weight when compared with the other BChE activity variables (total, absolute C5, and absolute C(OF)), its median activity level was used for the classification of CHE2 C5+ phenotypes (faint and intense). Mean body mass index (BMI) was compared among the CHE2 locus phenotypes-controlled by sex, age, and ethnic group. It was shown that the intense CHE2 C5+ phenotype presents a significantly lower (p < 0.001) mean BMI (23.2) than the other phenotypes (faint CHE2 C5+ = 25.2; CHE2 C5- = 25.4). It seems that the relative COF activity is positively associated with fat storage, since CHE2 C5- and faint CHE2 C5+ phenotypes showed higher mean BMI than the intense CHE2 C5+ phenotype. Our hypothesis is that the presence of C5 in a relatively high proportion leads to less fat storage.     

Molecular basis of the globoside-deficient P(k) blood group phenotype. Identification of four inactivating mutations in the UDP-N-acetylgalactosamine: globotriaosylceramide 3-beta-N-acetylgalactosaminyltransferase gene

The biochemistry and molecular genetics underlying the related carbohydrate blood group antigens P, P(k), and LKE in the GLOB collection and P1 in the P blood group system are complex and not fully understood. Individuals with the rare but clinically important erythrocyte phenotypes P(1)(k) and P(2)(k) lack the capability to synthesize P antigen identified as globoside, the cellular receptor for Parvo-B19 virus and some P-fimbriated Escherichia coli. As in the ABO system, naturally occurring antibodies, anti-P of the IgM and IgG class with hemolytic and cytotoxic capacity, are formed. To define the molecular basis of the P(k) phenotype we analyzed the full coding region of a candidate gene reported in 1998 as a member of the 3-beta-galactosyltransferase family but later shown to possess UDP-N-acetylgalactosamine:globotriaosylceramide 3-beta-N-acetylgalactosaminyltransferase or globoside synthase activity. Homozygosity for different nonsense mutations (C(202) --> T and 538insA) resulting in premature stop codons was found in blood samples from two individuals of the P(2)(k) phenotype. Two individuals with P(1)(k) and P(2)(k) phenotypes were homozygous for missense mutations causing amino acid substitutions (E266A or G271R) in a highly conserved region of the enzymatically active carboxyl-terminal domain in the transferase. We conclude that crucial mutations in the globoside synthase gene cause the P(k) phenotype.     

The haptoglobin-gene deletion responsible for anhaptoglobinemia.

We have found an allelic deletion of the haptoglobin (Hp) gene from an individual with anhaptoglobinemia. The Hp gene cluster consists of coding regions of the alpha chain and beta chain of the haptoglobin gene (Hp) and of the alpha chain and beta chain of the haptoglobin-related gene (Hpr), in tandem from the 5' side. Southern blot and PCR analyses have indicated that the individual with anhaptoglobinemia was homozygous for the gene deletion and that the gene deletion was included at least from the promoter region of Hp to Hpr alpha but not to Hpr beta (Hpdel). In addition, we found seven individuals with hypohaptoglobinemia in three families, and the genotypes of six of the seven individuals were found to be Hp2/Hpdel. The phenotypes and genotypes in one of these three families showed the father to be hypohaptoglobinemic (Hp2) and Hp2/Hpdel, the mother to be Hp2-1 and Hp1/Hp2, one of the two children to be hypohaptoglobinemic (Hp2) and Hp2/Hpdel, and the other child to be Hp1 and Hp1/Hpdel, showing an anomalous inheritance of Hp phenotypes in the child with Hp1. The Hp2/Hpdel individuals had an extremely low level of Hp (mean+/-SD = 0.049+/-0. 043 mg/ml; n=6), compared with the level (1.64+/-1.07 mg/ml) obtained from 52 healthy volunteers having phenotype Hp2, whereas the serum Hp level of an individual with Hp1/Hpdel was 0.50 mg/ml, which was approximately half the level of Hp in control sera from the Hp1 phenotype (1.26+/-0.33 mg/ml; n=9), showing a gene-dosage effect. The other allele (Hp2) of individuals with Hp2/Hpdel was found to have, in all exons, no mutation, by DNA sequencing. On the basis of the present study, the mechanism of anhaptoglobinemia and the mechanism of anomalous inheritance of Hp phenotypes were well explained. However, the mechanism of hypohaptoglobinemia remains unknown.     

Prolein polymorphism in a randombred chicken population

Polymorphism in plasma amylase, plasma alkaline phosphatase, non-specific esterase and red cell esterase-D of the Athens-Canadian randombred (ACRB) population of chickens was determined by polyacrylamide and starch gel electrophoresis. Amylase alleles Amy-1^A and Amy-1^B were segregating in the ACRB population with frequencies of 0.45 and 0.55 respectively. For the plasma alkaline phosphatase the F and S bands, the B band and a new isozyme migrating at a faster rate than the previously reported F band were detected. A genetic nomenclature for plasma alkaline phosphatase is suggested which considers the difference between the F and S bands as the presence or absence of sialic acid attached to a primary protein.
Plasma esterase activity was observed in all four of the regions previously reported, but there was no polymorphism found in any of the loci. All birds in this population showed the same red-cell esterase-D phenotype which consisted of a main band with sub-bands on each side.     

Differential enzyme activities in human esterase D phenotypes

Summary Red cell ESD activities have been determined in 78 individuals with ESD-1 phenotype, in 94 with ESD2-1 and in 28 with ESD-2 phenotype. The mean activities of these three groups were 276.7, 216.6 and 171.5 expressed as 10^-7 M methyl-umbelliferone produced/h/g Hb, respectively. The activities associated with a single ESD ^* 1 gene are estimated to be 60% higher than ESD ^* 2.     

Genotypic succession in the cyclomorphosis of Bosmina longirostris (Cladocera)

SUMMARY. The pattern of cyclomorphosis of Bosmina longirostris was followed over a 21-month period in Lake Travis, Texas. Appreciable variation was observed in body size and in the lengths of antennules and mucrones. Electrophoresis of clonal isolates collected over the period of study showed a seasonal cycle in esterase phenotypes. A definite association was demonstrated between the esterase phenotypes and morphologies of these clonal isolates. Individuals of one esterase phenotype were usually large and long-featured, whereas individuals of the other esterase phenotypes were smaller, with short features. The association of morphology and esterase phenotypes from laboratory experiments corresponded to the association observed in field samples. These results support the hypothesis that the cyclomorphosis of Lake Travis Bosmina involves a succession of clones as well as phenotypic plasticity.     

Quantification of plasticity of plant traits in response to light intensity: comparing phenotypes at a common weight

Summary Plasticity of plant traits is commonly quantified by comparing different phenotypes at the same age. In this paper, we present a method in which the effect of resource conditions on plant weight is used as a basis for quantifying the plasticity of individual plant traits. Abutilon theophrasti individuals were grown in, and some transferred between, high and low intensity light conditions, resulting in four phenotypes. Plant traits were found to exhibit different degrees of plasticity, decreasing in this order: height; specific leaf area; allocation to branch roots; allocation to leaf area; number of nodes; allocation to tap roots; allocation to stem; allocation to leaf weight. Under these conditions, individuals of the four phenotypes had very similar heights when compared at the same age, but very different heights when compared at the same plant weight. The latter comparison indicates that light intensity influences height independently of its influence on plant weight. Individuals that were transferred from high to low light had greater allocation that had not been transferred, but individuals of all phenotypes had nearly the same leaf weight allocation when compared at the same plant weight. The latter comparison indicates that light intensity influeces leaf weight allocation mostly by influencing plant weight. In the phenotype resulting from the transfer of plants from low to high light, reproduction was stimulated much less than plant weight and axillary leaf growth, and reproductive allocation was delayed relative to the other three phenotypes. We conclude that when plasticity is measured by comparing phenotypes at the same plant weight, the effects of resources on plant size can be excluded from the quantification.     

Hydroxylation of CMP-NeuAc controls the expression of N-glycolylneuraminic acid in GM3 ganglioside of the small intestine of inbred rats

An enzymatic activity responsible for the hydroxylation of CMP-NeuAc into CMP-N-glycolylneuraminic acid (CMP-NeuGc) was found in the cytosolic fraction after cellular fractionation of the mucosa of rat small intestine. It was maximum in the presence of NADPH or NADH, but it was reduced by 50% by addition of 1 mM EDTA. The Km value for CMP-NeuAc was 0.6 microM. The CMP-NeuAc hydroxylase activity paralleled the expression of the GM3 (NeuGc) phenotype in the epithelium of the small intestine and was not measurable in the mutant rats BN and SHR that only expressed GM3 (NeuAc). Furthermore, the only form of CMP-sialic acid present in the intestinal mucosa of the mutants was CMP-NeuAc, whereas in the other strains CMP-NeuGc accounted for 70-85% of the native CMP-sialic acids. Wild-type and CMP-NeuAc hydroxylase-deficient inbred rats were mated. Individuals of F1 and backcross generations were typed for the phenotypes GM3(NeuGc)/GM3(NeuAc) and the activity of CMP-NeuAc hydroxylase in the small intestine. It was found that the expression of NeuGc in GM3 depends on a single autosomal dominant gene and correlates with the activity of CMP-NeuAc hydroxylase. Two tissues other than small intestine, kidney and spleen, which expressed GM3(NeuGc) in BN and SHR, also expressed the CMP-NeuAc hydroxylase activity, as in the other strains. It was concluded that the key enzyme responsible for the presence of NeuGc in GM3 is a CMP-NeuAc hydroxylase and that mutant rats carry a defect that is specific to intestine. The comparative analysis of the respective contribution of NeuGc and NeuAc to the glycoprotein sialic acids of the small intestine showed that CMP-NeuAc hydroxylase is also responsible for part of the NeuGc present in the glycoproteins. However, the occurrence of 20-30% of NeuGc in the intestinal glycoproteins of the CMP-NeuAc hydroxylase-deficient rats indicated that there is another enzyme providing intestinal glycoproteins with NeuGc and operating under a different genetic control.     

Red cell enzyme polymorphisms in three Iranian population samples: Tabriz,Yazd, Mashhad

Three population samples from Iran (Tabriz, Yazd, Mashhad) have been typed for four enzyme group polymorphisms: ACP1, ESD, AK, and PGD. The phenotype and allele frequencies are presented and compared with other Iranian populations. The AK allele frequencies do not show significant intergroup heterogeneity, whereas ACP1, ESD and PGD allele frequencies disclose obvious heterogeneity. The possible reasons therefore are discussed.