The gene for human peptidase A is on band 18q23 and shows triplex and uniplex dosage effect

Summary Gene dosage effect for the enzyme peptidase A was studied in the red cells of subjects trisomic (seven cases) or monosomic (five cases) for the segment of chromosome 18 carrying the gene. The individual levels of enzyme activity in both groups were different from those of the controls, but with a wide overlap. The use of the ratio of the activity of each subject to the midparent activity eliminated the overlapping. The mean ratio was 0.94 for the controls, 2.36 for the trisomics, and 0.41 for the monosomics. The trisomic ratio is higher than expected on the assumption of a linear effect. Correlation with the cytogenetic data in four cases of ring 18 and one of 18q-firmly places the gene for peptidase A on band 18q23.     

Types of enzymatic overdosing in trisomy 21: erythrocytic superoxide dismutase-AJ and phosphoglucomutase.

A comparative study was carried out on the hemolysates of 6 trisomic 21 and 6 normal subjects, by electrophoresis in starch gel, determining by a combined staining method both SOD-A (former IPO-dimer) and PGM activity. The enzymes were found statistically to be in a hyperactive status, the ratio of trisomic to normal values being approximately equalt to 1.4. SOD-A supraactivation is the effect of a genic dose, as demonstrated in earlier works (Sichitiu, 1973; Sichitiu et al., 1974; Sinet et al., 1974), whereas PGM hyperactivity appears to be modified secondarily, the same as the activity of other cellular enzymes in Down's disease.     

Regional assignment of catalase (CAT) gene to band 11p13. Association with the aniridia-Wilms' tumor-Gonadoblastoma (WAGR) complex

A gene dosage effect for catalase (CAT) was investigated in three individuals : one with 11p13 deletion, aniridia, ambiguous genitalla, and gonadoblastoma ; one trisomic for 11p with the exception of 11p13; and one trisomic for 11p13. Results were compatible with the assignment of CAT to 11p13 and its linkage with the aniridia-gonadoblastoma or Wilms' tumor complex (WAGR).     

Analysis of Autosomal Dosage Compensation Involving the Alcohol Dehydrogenase Locus in Drosophila Melanogaster

An example of autosomal dosage compensation involving the expression of the alcohol dehydrogenase (Adh) locus is described. Flies trisomic for a quarter of the length of the left arm of chromosome two, including Adh, have diploid levels of enzyme activity and alcohol dehydrogenase messenger RNA. Subdivision of the compensating trisomic into smaller ones revealed a region that exerts an inverse regulatory effect on alcohol dehydrogenase activity and messenger RNA levels and a smaller region surrounding the structural gene that exhibits a direct gene dosage response. The two opposing effects are of sufficient magnitude that they cancel when simultaneously present resulting in the observed compensation in the larger aneuploid. An Adh promoter-white structural gene fusion construct is affected by the inverse regulatory region indicating that the effect is mediated through the Adh promoter sequences. The role of autosomal dosage compensation in understanding aneuploid syndromes and karyotype evolution in Drosophila species is discussed.     

Direct gene dosage determination in patients with unbalanced chromosomal aberrations using cloned DNA sequences. Application to the regional assignment of the gene for alpha 2(I) procollagen (COLIA2).

We describe a new method of direct gene dosage determination in patients with unbalanced chromosomal aberrations using cloned DNA sequences: the intensity of the signal obtained by hybridization of the radioactive probe to the corresponding DNA fragments can be compared with the intensity of the DNA fragments that hybridize with a nonsyntenic probe used as an internal control. This has been demonstrated by densitometer tracing of the autoradiogram, using an X-specific DNA sequence, beta globin and alpha 2(I) collagen probes, in normal men and women, in one patient trisomic for 11p, and in one patient trisomic for segment 7q21 leads to 7qter. The ratio men/women for the X-specific sequence (DXS) was close to the expected value 0.5, while the ratio trisomy 11/normal control and trisomy 7/normal control were close to 1.5 for beta globin (HBB) and alpha 2(I) collagen (COLIA2), respectively. The gene coding for COLIA2 can therefore be assigned to 7q21 leads to 7qter. This method should also apply to noncoding sequences: the increasing number of cloned DNA segments that have already been assigned to a specific chromosome represent a new tool for prenatal and premorbid diagnosis of unbalanced chromosomal aberrations.     

Dosage effects for superoxide dismutase-1 in nucleated cells aneuploid for chromosome 21.

Assays of the activity of chromosome 21 determined superoxide dismutase-1 (SOD-1) in lymphocytes and polymorphonuclear granulocytes have demonstrated 38% and 40% increases, respectively, in cells from individuals with trisomy 21. Similarly, SOD-1 activity in trisomic fibroblasts is increased by 81%, while cells monosomic for chromosome 21 have only 60% of normal activity. Taken together with the data on SOD-1 activities in trisomic erythrocytes and platelets, the present results firmly confirm the existence of a true dosage effect for this enzyme in cells aneuploid for chromosome 21. However, the results of assays of the activity of glutathione peroxidase in trisomic fibroblasts did not confirm the possibility previously reported of a chromosome 21 related dosage effect for this enzyme.     

Involvement of glutathione and glutathione-related enzymes in the protection of normal and trisomic human fibroblasts against daunorubicin

We measured the glutathione content, and the activity of glutathione-related enzymes and DT-diaphorase in cultured normal (cell line: S-126) and trisomic (cell lines: S-158, S-240) human fibroblasts exposed to daunorubicin (DNR). Determination of reduced and total glutathione levels, and measurement of the activity of glutathione peroxidase, glutathione reductase, glutathione-S-transferase and DT-diaphorase were performed spectrophotometrically. Human fibroblasts were exposed to 4 microm DNR for 2 h, and the cells placed in drug-free medium for 6, 12, 24, 48, and 72 h. Cellular levels of GSH and total glutathione decreased following exposure to DNR. However, the ratio of GSH to total glutathione returned to control levels only in trisomic cells. These changes were concomitant with increasing glutathione-S-transferase and glutathione reductase activities. DNR also significantly increased the activity of Se-independent peroxidase and DT-diaphorase in trisomic fibroblasts. Marked increases in the activity of Se-dependent peroxidase and DT-diaphorase alone were seen in normal cells. The results provide the first evidence that DNR can induce alterations in the level of glutathione and glutathione-dependent enzymes in trisomic fibroblasts as compared to normal cells, which may provide additional protection against daunorubicin-induced oxidative stress in trisomic fibroblasts.     

Preliminary characterization of a Mg2+-ATPase in hamster sperm head membranes

The gene, IFRC, on human chromosome 21 has been considered, on the basis of indirect functional and antibody blocking evidence, to code for an interferon receptor. To obtain direct evidence for this conclusion, the specific binding of ¹²⁵I-HuIFN-αA to matched sets of fibroblasts diploid and aneuploid for chromosome 21 has been determined. Although the dissociation constant for IFN-α is the same for both trisomic and diploid cells, trisomic cells bind more IFN-α and monosomic cells less than do diploid cells. When the data for all determinations are combined, the monosomy 21:diploid:trisomy 21 binding ratios are 0.5:0.8:1.5, in good agreement with the values of 0.5:1.0:1.5 expected on the basis of strict gene dosage considerations for a product coded for by chromosome 21. We conclude, therefore, that the chromosome 21 gene product determined by IFRC and recognized by antibodies which block interferon action is truly a specific cell surface receptor for human interferon-α.     

Enzyme patterns in trisomy 19 of the mouse

Activity patterns of cytosolic and mitochondrial enzymes of carbohydrate and amino acid metabolism have been measured in murine trisomy 19. In spite of marked hypoplasia, no significant alterations of the patterns (per gram of organ weight) were observed, with the exception of glutamate oxaloacetate transaminase (GOT-1), and phosphoglycerate mutase (PGAM). Clear-cut gene dosage effects in liver, brain, heart, skeletal muscle, and erythrocytes of fetal and newborn mice, confirm the assignment of GOT-1 to chromosome 19. Data obtained for PGAM demonstrate that one of the two different subunits leading to organ-specific isozyme patterns of the dimer enzyme protein is coded on chromosome 19 (gene Pgam-1). Dosage effects are fully expressed in liver, brain, and erythrocytes (AA-type isozyme), but not in skeletal muscle (BB-type isozyme). Dosage effects on the hybrid AA-AB-BB-isozyme pattern in the course of development of the heart muscle, were demonstrated by means of quantitative activity measurement after electrophoretic separation. The comparison of enzyme patterns of eusomic and trisomic erythrocytes, produced after injection of fetal stem cells into irradiated adult carriers (transplantation chimaeras), revealed enzyme activity ratios that were similar to those produced by erythrocytes of adult euploid and trisomic mice. This is in agreement with the chromosome assignments and dosage effects mentioned above.     

Cystathionine beta synthase: gene dosage effect in trisomy 21

The enzymatic activity of cystathionine beta synthase has been studied in fibroblasts of nine patients with regular trisomy 21. An excess of CBS activity was found in trisomy 21 with a trisomy 21/normal ratio equal to 1.66. A 1.04 ratio was found in 21q21----21 p ter monosomy; a 1.04 and 0.99 ratio was found in two 21 qter----21q22.3 monosomies; a 1.14 ratio in 21 qter----21q22 monosomy; a 0.89 ratio in a 21q21----21 pter trisomy; an excess of CBS activity was found in a 21q22.1 ----21q21 trisomy with a 1.57 ratio. These results show a gene dosage effect in human fibroblasts trisomic for chromosome 21 and suggest the assignment of human CBS locus between 21q22.1 and 21q21.     

Chromosomal and enzymatic patterns provide evidence for two types of human colon cancers with abnormal nucleotide metabolism

Correlated chromosomal and enzymatic studies are reported in a series of 13 human colon cancers. Two distinct chromosomal patterns are found: one with many losses, called monosomic type and another with many gains, called trixomic type. Chromosome 18, which carries the gene for thymidylate synthase (TS) is always missing in the monosomic type, in which TS activity is low. On the contrary, TS activity is high in trysomic type on the average. The long arm of chromosome 17, which carries the gene for thymidine kinase (TK), is frequently duplicated in both monosomic and trisomic and trisomic types, in which TK activities are high. It is found that most other deletions affect chromosomes carrying genes for enzymes of the de novo pathways whereas most other gains affect chromosomes carrying for enzymes of salvage pathways for synthesis of nucleotides. The importance of this finding is discussed.     

Effects of increased major histocompatibility complex dosage on chicken monocyte-macrophage function

The influence of major histocompatibility complex (B complex) dosage on monocyte-macrophage function was examined using 4- to 6-week-old trisomic strain chickens. Di- (B15B15), tri- (B15B15B15), and tetrasomic (B15B15B15B15) progeny were produced from trisomic x trisomic crosses. Although mononuclear leukocytes from tetrasomics exhibited enhanced chemotactic activity in response to both f-met-leu-phe and Enterobacter cloacae culture supernatant as compared with that of cells from other groups, the ability to generate peritoneal exudate cells in response to intraperitoneal Sephadex stimulation was similar in all groups. Among peritoneal exudate cells, tetrasomic birds produced a significantly lower percentage of adherent macrophages with a higher proportion of Fc receptor-positive and CMTD-2-reactive macrophages than either disomic or trisomic chickens. Both tetrasomic and trisomic peritoneal macrophages exhibited a reduced phagocytic activity for unopsonized but not opsonized SRBC than was found with disomic macrophages. Thus, the number of major histocompatibility complex copies present in cells appears to influence monocyte-macrophage function.     

Self-cloning in Streptomyces griseus of an str gene cluster for streptomycin biosynthesis and streptomycin resistance.

An str gene cluster containing at least four genes (strR, strA, strB, and strC) involved in streptomycin biosynthesis or streptomycin resistance or both was self-cloned in Streptomyces griseus by using plasmid pOA154. The strA gene was verified to encode streptomycin 6-phosphotransferase, a streptomycin resistance factor in S. griseus, by examining the gene product expressed in Escherichia coli. The other three genes were determined by complementation tests with streptomycin-nonproducing mutants whose biochemical lesions were clearly identified. strR complemented streptomycin-sensitive mutant SM196 which exhibited impaired activity of both streptomycin 6-phosphotransferase and amidinotransferase (one of the streptomycin biosynthetic enzymes) due to a regulatory mutation; strB complemented strain SD141, which was specifically deficient in amidinotransferase; and strC complemented strain SD245, which was deficient in linkage between streptidine 6-phosphate and dihydrostreptose. By deletion analysis of plasmids with appropriate restriction endonucleases, the order of the four genes was determined to be strR-strA-strB-strC. Transformation of S. griseus with plasmids carrying both strR and strB genes enhanced amidinotransferase activity in the transformed cells. Based on the gene dosage effect and the biological characteristics of the mutants complemented by strR and strB, it was concluded that strB encodes amidinotransferase and strR encodes a positive effector required for the full expression of strA and strB genes. Furthermore, it was found that amplification of a specific 0.7-kilobase region of the cloned DNA on a plasmid inhibited streptomycin biosynthesis of the transformants. This DNA region might contain a regulatory apparatus that participates in the control of streptomycin biosynthesis.     

Association of the yellow leaf (y10) mutant to soybean chromosome 3

At least 19 single recessive gene yellow leaf mutants and one duplicate recessive gene mutant have been described in soybean. This study was conducted to associate a yellow leaf mutant, y10, with a specific soybean chromosome by using primary trisomics (2n = 41). Seven soybean primary trisomics were hybridized as female parent with genetic stock strain, T161, carrying y10. F(1) disomic and primary trisomic plants were identified cytologically. One disomic (control) and all primary trisomic plants were allowed to self-pollinate and F(2) populations were classified for green versus yellow leaf mutant. The F(2) population of Triplo 3 segregated in a 17:1 ratio, while a disomic (3:1) ratio was observed with Triplo 8-, 17-, 18-, and 20-derived F(2) populations, suggesting that the y10 locus is on chromosome 3. The y10 locus was examined with four simple sequence repeat (SSR) markers (Satt584, Sat_033, Satt387, and Satt022) from molecular linkage group (MLG) N and y10 was found linked with Satt022. Therefore we confirmed the association of MLG N with chromosome 3. The possible association of y10 with Triplo 16 and Triplo 19 are discussed.     

Neurochemical Changes in Murine Trisomy 16: Delay in Cholinergic and Catecholaminergic Systems

Abstract: Two strains of Mus musculus musculus , C57BL/6J and CD-1, and Mus musculus poschiavinus , the tobacco mouse, were used to study the effects of increased gene dosage of mouse chromosome 16 (MMU 16). A developmental delay has been found in the brains of murine trisomy 16 (Ts 16) fetuses. Both the brain weight (in all three strains) and DNA content (in CD-1) were reduced, while protein content was unchanged in Ts16 compared to normal littermates. The daily increments of weight and protein (except in M. m. poschiavinus ) were significantly greater in Ts16. The activities of choline acetyltransferase and acetylcholinesterase and nuscarinic receptor binding were reduced. Their daily increments were also reduced to less than 56% that of littermates in Ts16 brains. The rate limiting enzymes of Catecholaminergic neurons, tyrosine hydroxylase and do-pamine β-hydroxylase, and the concentration of catecholamines in the brains of Ts16 animals were lower. The activities of three other Catecholaminergic enzymes, DOPA decarboxylase, catechol O -methyltransferase, and monoamine oxidase, were generally elevated in Ts16 brain, as were their daily increments. These observations indicate a significant developmental alteration in the maturation of the trisomic brain and suggest future avenues for defining the effect of increased gene dosage of MMU 16 in the CNS.     

Massively Parallel Sequencing Reveals the Complex Structure of an Irradiated Human Chromosome on a Mouse Background in the Tc1 Model of Down Syndrome

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype – phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.     

Isolation of Escherichia coli mutants with elevated levels of membrane enzymes. A trans-acting mutation controlling diglyceride kinase

We have developed a rapid autoradiographic colony assay for detecting mutants with elevated levels of certain biosynthetic enzymes. Four Escherichia coli strains in which the specific activity of the membrane enzyme diglyceride kinase is increased 5-10-fold have been obtained with this approach. The mutant kinase has the same thermal denaturation profile and subcellular localization as the wild type. Five other membrane enzymes involved in phospholipid bilayer assembly are unaffected. In one of these strains (GK-1) the mutation (dgkR-1) responsible for the elevated kinase has been mapped at a new site near minute 92, while the previously identified structural gene (dgk) lies near minute 90. When the structural gene for the kinase (dgk) is cloned on a multi-copy vector-like ColE1, the kinase can be overproduced 5-10-fold on the basis of gene dosage (Lightner, V. A., Larson, T. J., Tailleur, P., Kantor, G. D., Raetz, C. R. H., Bell, R. M., and Modrich, P. (1980) J. Biol. Chem. 255, 9413-9420). Introduction of such hybrid plasmids into a mutant harboring dgkR-1 leads to a multiplicative (rather than additive) effect, resulting in specific activities of diglyceride kinase that are 35-75-fold higher than normal. These results show that dgkR-1 is a trans-acting mutation and suggest the existence of novel regulatory proteins (or metabolites) that direct the expression of certain membrane enzymes.     

Regulation of beta-glucuronidase synthesis in Escherichia coli K-12: pleiotropic constitutive mutations affecting uxu and uidA expression.

Among the beta-glucuronidase (UID)-constitutive mutants obtained by growth on methyl-beta-D-galacturonide, some strains are also derepressed for the two enzymes of the uxu operon: mannonate oxidoreductase (MOR) and mannonate hydrolyase (HLM). By conjugation and transduction experiments, two distinct constitutive mutations were separated in each pleiotropic mutant strain. One of them was specific for uidA gene expression and was characterized as affecting either uidO or uidR sites. The second type of mutation was mapped close to the uxu operon and was found to be responsible for the pleiotropic effect revealed in the primary mutants: after separation such a mutation still fully derepresses MOR and HLM synthesis but weakly derepresses UID synthesis. The pleiotropic effect of this mutation was maintained even though the activity of the structural genes was altered. This rules out the occurrence of an internal derepressing interaction between these enzymes. In merodiploid strains, uxu-linked constitutive mutations were recessive to the wild-type allele, suggesting that these mutations could affect a regulatory gene. The uxuR gene is probably a specific regulatory gene for a very close operon, uxu. Moreover, it has a weak effect on uidA expression. Thus, UID synthesis would be negatively controlled through the activity of two repressor molecules that are synthesized by two distinct regulatory genes, uidR and uxuR. These two repressing factors are antagonized, respectively, by phenyl-thio-beta-D-glucuronide and mannonic amide and could cooperate in a unique repression/induction control over uidA expression. Constitutive mutations affecting the control sites of uidA gene probably characterize two distinct attachment sites in the operator locus for each of the repressor molecules.     

PK3: A new chromosome enzyme marker for gene dosage studies in chromosome 15 imbalance

Summary Gene dosage studies yielded results consistent with the assignment of the locus for pyruvate kinase (PK 3) to chromosome 15. The activity of seven cytoplasmic enzymes has been determined in fibroblast extracts from six trisomy 15 lines and 16 normal control lines. The fibroblast extracts from the trisomic patients had pyruvate kinase activity 57% higher than fibroblast extracts from control lines, while other enzyme activities were within the normal range of activity.     

Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae

Summary The galactose analogue 2-deoxygalactose was found to inhibit the growth of a mutant strain of Saccharomyces cerevisiae constitutively producing the set of galactose utilization enzymes. Based on this fact, the yeast GAL80 gene negatively regulating the expression of the genes encoding those enzymes was isolated for its ability to confer 2-deoxygalactose resistance on a strain carrying a recessive mutation in that gene. The GAL80 gene was located within a 3.0 kb fragment in the cloned DNA. When the isolated gene was incorporated into a multi-copy plasmid, the induced level of three enzymes encoded by the gene cluster GAL7-GAL10-GAL1 in the host chromosome was lowered. Such a gene dosage effect of GAL80 was further pronounced if sucrose, a sugar causing catabolite repression, was added to the growth medium. The ratio of the enzyme activity of the yeast bearing multiple copies of GAL80 to that of the yeast bearing its single copy significantly varied with the enzyme. From these results we suggest that the intracellular inducer interacts with the GAL80 product and that GAL80 molecules directly bind the GAL cluster genes with an affinity different from one gene to another.The first article of this series is in Mol Gen Genet 191:31–38On a leave absence from Nikka Whisky Co.