A locus regulating N-acetylglucosaminidase synthesis during development in dictyostelium

The cellular specific activity of N-acetylglucosaminidase increases during development in Dictyostelium discoideum. A monoclonal antibody which specifically recognizes Mr 68,000 and 67,000 forms of N-acetylglucosaminidase was used to show that changes in the relative rate of enzyme synthesis during development parallel the pattern of enzyme accumulation. Developmental and regulatory mutants were isolated to study the relationship between development and enzyme accumulation. No evidence was obtained for any dependence of enzyme accumulation on those genes that are required for aggregation. However, a separate regulatory locus was identified which is involved in enzyme accumulation. Mutations in this gene, nagC, prevent enzyme accumulation during development by preventing an increase in the relative synthetic rate of N-acetylglucosaminidase. The accumulation of other enzymes is unaffected and the mutation causes no developmental defects other than those caused by the loss of N-acetylglucosaminidase activity. The nagC mutation, which is recessive, maps to linkage group VI and is therefore unlinked to the structural gene for N-acetylglucosaminidase.     

A β-glucosidase gene of Dictyostelium discoideum

Seven mutations affecting β-glucosidase activity in Dictyostelium discoideum were found to be non-complementing, recessive to the wild-type allele, and to occur in the gene locus, gluA. This gene, which is likely to be the structural gene for β-glucosidase, since a mutation in it gives rise to thermolabile activity and other mutations in it result in no measurable activity, was mapped to linkage group VI. The expression of the β-glucosidase gene is regulated such that the enzyme is synthesized during the growth phase and during culmination, but not during the first 18 hours following the initiation of development. If expression of the structural gene required the function of a positive regulatory protein coded for by a gene as mutable as the gluA gene, there was greater than 99% chance one of the mutations of this series would have affected the regulatory locus. The absence of a second complementing locus for β-glucosidase suggests that this enzyme is regulated by other means.     

Purification, characterization and gene analysis of N-acetylglucosaminidase from Enterobacter sp. G-1.

Enterobacter sp. G-1 is a bacterium isolated previously as a chitinase-producing bacterium. We found this bacterium also produced N-acetylglucosaminidase and characterized that in this study. Extracellular N-acetylglucosaminidase of 92.0 kDa was purified near homogeneity by 8.57-fold from Enterobacter sp. G-1. The optimum temperature and the optimum pH of the purified N-acetylglucosaminidase was 45 degrees C and 6.0, respectively. The N-terminal amino acid sequence of 23 residues of N-acetylglucosaminidase was identified. Based on the N-terminal sequence, we amplified pieces of the DNA fragments by PCR. Using these PCR products as probes, we screened the genomic library and successfully isolated the entire N-acetylglucosaminidase gene (designated nag1) from Enterobacter sp. G-1. The nucleotide sequence of the nag1 gene was found to consist of 2,655 bp encoding a protein of 885 amino acid residues. Comparison of the deduced amino acid sequence from the nag1 gene found 97.3% identity with chitobiase from Serratia marcescens, 54.4% identity with N,N'-diacetylchitobiase from Vibrio harveyi, and 42.7% identity with N-acetylglucosaminidase (ExoI) from Vibrio furnissii. Enzymatic activity assay of N-acetylglucosaminidase indicated stronger activity toward PNP-GlcNAc than PNP-(GlcNAc)2 or PNP-(GlcNAc)3.     

Genetic characterization and regulation of the nadB locus of Salmonella typhimurium.

The nadB locus encodes the first enzyme of NAD synthesis. It has been reported that this gene and nadA are regulated by a positive regulatory protein encoded in the nadB region. In pursuing this regulatory mechanism, we constructed a fine-structure genetic map of the nadB gene. The region appears to include a single complementation group; no evidence for a positive regulatory element was found. Several mutations causing resistance to the analog 6-aminonicotinamide mapped within the structural gene and probably cause resistance to feedback inhibition. Regulatory mutations for nadB were isolated. These mutants mapped far from nadB near the pnuA gene, which encodes a function required for nicotinamide mononucleotide transport. The regulatory mutations appear to affect a distinct function encoded in the same operon as pnuA.     

Structural Analysis of the dur Loci in S. CEREVISIAE: Two Domains of a Single Multifunctional Gene

In Saccharomyces cerevisiae, the degradation of urea to carbon dioxide and ammonia is catalyzed by urea carboxylase and allophanate hydrolase. The loci coding for these enzymes (dur1 and dur2) are very tightly linked on the right arm of chromosome II between pet11 and met8. Pleiotropic mutations that fail to complement mutations in either of the dur loci were found to be predominantly located in or near the dur2 locus. We interpret these data as suggesting that the two dur loci might in reality be domains of a single gene that codes for a multifunctional polypeptide. In view of this conclusion, we have renamed the dur loci as the dur1,2 locus.     

Heritable targeted mutagenesis in maize using a designed endonuclease

The liguleless locus ( liguleless1 ) was chosen for demonstration of targeted mutagenesis in maize using an engineered endonuclease derived from the I- Cre I homing endonuclease. A single-chain endonuclease, comprising a pair of I- Cre I monomers fused into a single polypeptide, was designed to recognize a target sequence adjacent to the LIGULELESS1 ( LG1 ) gene promoter. The endonuclease gene was delivered to maize cells by Agrobacterium -mediated transformation of immature embryos, and transgenic T₀ plants were screened for mutations introduced at the liguleless1 locus. We found mutations at the target locus in 3% of the T₀ plants, each of which was regenerated from independently selected callus. Plants that were monoallelic, biallelic and chimeric for mutations at the liguleless1 locus were found. Relatively short deletions (shortest 2 bp, longest 220 bp) were most frequently identified at the expected cut site, although short insertions were also detected at this site. We show that rational re-design of an endonuclease can produce a functional enzyme capable of introducing double-strand breaks at selected chromosomal loci. In combination with DNA repair mechanisms, the system produces targeted mutations with sufficient frequency that dedicated selection for such mutations is not required. Re-designed homing endonucleases are a useful molecular tool for introducing targeted mutations in a living organism, specifically a maize plant.     

Rufous oculocutaneous albinism in southern African Blacks is caused by mutations in the TYRP1 gene.

Oculocutaneous albinism (OCA) is the most common autosomal recessive disorder among southern African Blacks. There are three forms that account for almost all OCA types in this region. Tyrosinase-positive OCA (OCA2), which is the most common, affects approximately 1/3,900 newborns and has a carrier frequency of approximately 1/33. It is caused by mutations in the P gene on chromosome 15. Brown OCA (BOCA) and rufous OCA (ROCA) account for the majority of the remaining phenotypes. The prevalence of BOCA is unknown, but for ROCA it is approximately 1/8,500. Linkage analysis performed on nine ROCA families showed that ROCA was linked to an intragenic marker at the TYRP1 locus (maximum LOD score = 3.80 at straight theta=.00). Mutation analysis of 19 unrelated ROCA individuals revealed a nonsense mutation at codon 166 (S166X) in 17 (45%) of 38 ROCA chromosomes, and a second mutation (368delA) was found in an additional 19 (50%) of 38 chromosomes; mutations were not identified in the remaining 2 ROCA chromosomes. In one family, two siblings with a phenotypically unclassified form of albinism were found to be compound heterozygotes for mutations (S166X/368delA) at the TYRP1 locus and were heterozygous for a common 2.7-kb deletion in the P gene. These findings have highlighted the influence of genetic background on phenotype, in which the genotype at one locus can be influenced by the genotype at a second locus, leading to a modified phenotype. ROCA, which in southern African Blacks is caused by mutations in the TYRP1 gene, therefore should be referred to as "OCA3," since this is the third locus that has been shown to cause an OCA phenotype in humans.     

Isolation and Mapping of Phosphotransferase Mutants in Escherichia coli

Mutants of Escherichia coli K-12 defective in enzyme I or Hpr, the two common components of the phosphoenolpyruvate-dependent phosphotransferase system, were isolated by a simple, direct method. The ptsI locus, the structural gene for enzyme I, and the ptsH locus, the site of mutations leading to loss of Hpr activity, are adjacent genes and could be part of a single operon. These two genes lie between the purC and supN markers in the order: strA... guaB-purC-ptsI-ptsH-supN-dsdA... his.     

The regulation of''early'' enzymes during the development and dedifferentiation of Dictyostelium discoideum

The specific activities of the enzymes alpha-mannosidase and N-acetylglucosaminidase increase immediately after the initiation of the development of bacterially grown cell cultures of Dictyostelium discoideum. The regulation of these two enzymes was found to be dissociable in the developmental timer mutant, FM-1, which aggregates 4.5 h earlier than wild-type cells due to the absence of the first rate-limiting component of the preaggregative period. The increase in alpha-mannosidase activity occurs in the absence of the first rate-limiting component, but the increase in N-acetylglucosaminidase activity does not. These results indicate the following: (1) the increase in the specific activity of alpha-mannosidase is not related to the timing of subsequent developmental stages; (2) the increase in the specific activity of N-acetylglucosaminidase is not necessary for the subsequent developmental program; and (3) either the increase in the specific activity of N-acetylglucosaminidase is dependent upon progress through the first rate-limiting component, or the increase in this enzyme activity and the first rate-limiting component are both dependent upon an early event for which FM-1 is defective. In addition to early development, we monitored the two enzyme activities during dedifferentiation. The results demonstrate that there is no difference between dedifferentiating wild-type cells and dedifferentiation-defective mutant HI-4 cells. Changes in enzyme specific activity accompanying dedifferentiation are dependent upon the composition of the dedifferentiation-inducing media and are consistent with the levels of these enzymes observed in cells growing in the different nutrient media.     

A Gene Controlling Variation in Arabidopsis Glucosinolate Composition Is Part of the Methionine Chain Elongation Pathway

Arabidopsis and other Brassicaceae produce an enormous diversity of aliphatic glucosinolates, a group of methionine (Met)-derived plant secondary compounds containing a beta-thio-glucose moiety, a sulfonated oxime, and a variable side chain. We fine-scale mapped GSL-ELONG, a locus controlling variation in the side-chain length of aliphatic glucosinolates. Within this locus, a polymorphic gene was identified that determines whether Met is extended predominantly by either one or by two methylene groups to produce aliphatic glucosinolates with either three- or four-carbon side chains. Two allelic mutants deficient in four-carbon side-chain glucosinolates were shown to contain independent missense mutations within this gene. In cell-free enzyme assays, a heterologously expressed cDNA from this locus was capable of condensing 2-oxo-4-methylthiobutanoic acid with acetyl-coenzyme A, the initial reaction in Met chain elongation. The gene methylthioalkylmalate synthase1 (MAM1) is a member of a gene family sharing approximately 60% amino acid sequence similarity with 2-isopropylmalate synthase, an enzyme of leucine biosynthesis that condenses 2-oxo-3-methylbutanoate with acetyl-coenzyme A.     

Two mutations within the coding sequence of the phenylalanine hydroxylase gene

Summary Two previously unidentified mutations at the phenylalanine hydroxylase locus were found during a study of the relationship between genotype and phenotype in phenylketonuria and hyperphenylalaninemia. One mutation eliminates the BamHI site in exon 7 and the other eliminates the HindIII site in exon 11 of the phenylalanine hydroxylase gene. They were suspected because of deviating restriction fragment patterns and confirmed by amplification, via the polymerase chain reaction, of exon 7 and exon 11, respectively, followed by digestion with the appropriate restriction enzyme. Direct sequencing of amplified mutant exon 7 revealed a G/C to T/A transversion at the first base of codon 272, substituting a GGA glycine codon for a UGA stop codon. Direct sequencing of amplified mutant exon 11 revealed a deletion of codon 364, a CTT leucine codon. The exon 7 mutation can be expected to result in a truncated protein and the exon 11 mutation in the elimination of an amino acid in the catalytic region of the enzyme. A patient who is a compound heterozygote for these two mutations has classical phenylketonuria. It is concluded that each of the two mutations leads to a profound loss of enzymatic activity. The segregation of these mutations with disease alleles in 4 and 2 families, respectively, supports the hypothesis that multiple mutations at the phenylalanine hydroxylase locus explain the variable phenylalanine tolerance in patients with phenylalanine hydroxylase deficiency.     

Genetic Analysis of the Cha-1-Unc-17 Gene Complex in Caenorhabditis

In C. elegans, the gene cha-1 is the structural gene for choline acetyltransferase, the enzyme which synthesizes acetylcholine. cha-1 is a complex gene which includes the previously described unc-17 locus; it has been hypothesized that a single protein is encoded which consists of several discrete structural domains. Mutations of the cha-1-unc-17 locus can be assigned to one of four classes on the basis of phenotype and complementation properties. A fine-structure map of this region has now been obtained by recombinational mapping. It is a large locus, spanning at least 0.035 map unit. On the map, the mutations lie in four contiguous, nonoverlapping regions, corresponding exactly to the different classes as defined by complementation and phenotype. Several new cha-1 mutations are described and mapped in the present study, including temperature-sensitive and lethal alleles.     

GDF5 is a second locus for multiple-synostosis syndrome

Multiple-synostosis syndrome is an autosomal dominant disorder characterized by progressive symphalangism, carpal/tarsal fusions, deafness, and mild facial dysmorphism. Heterozygosity for functional null mutations in the NOGGIN gene has been shown to be responsible for the disorder. However, in a cohort of six probands with multiple-synostosis syndrome, only one was found to be heterozygous for a NOGGIN mutation (W205X). Linkage studies involving the four-generation family of one of the mutation-negative patients excluded the NOGGIN locus, providing genetic evidence of locus heterogeneity. In this family, polymorphic markers flanking the GDF5 locus were found to cosegregate with the disease, and sequence analysis demonstrated that affected individuals in the family were heterozygous for a novel missense mutation that predicts an R438L substitution in the GDF5 protein. Unlike mutations that lead to haploinsufficiency for GDF5 and produce brachydactyly C, the protein encoded by the multiple-synostosis-syndrome allele was secreted as a mature GDF5 dimer. These data establish locus heterogeneity in multiple-synostosis syndrome and demonstrate that the disorder can result from mutations in either the NOGGIN or the GDF5 gene.     

Arg-7 mutant x wild-type crosses in Chlamydomonas reinhardi: Study of the enzyme produced in diploid strains

Summary The arg-7 locus is the structural gene for the argininosuccinate lyase (ASL). Interallelic complementation was previously found to occur between several mutants of the locus: this is indicative for the homomultimeric nature of ASL.Two complementing (arg-7-5 and arg-7-7) and two non-complementing (arg-7-1 and arg-7-6) mutants of the arg-7 locus were crossed to the pab-2 strain (which is wild-type for the arg-7 locus). In each cross, heterozygote phenotypically wild-type strains were isolated; their diploid pattern was demonstrated by various criteria: mating type, cell volume, nuclear size.The four heterozygotes were compared to the haploid wild-type and in some experiments, to the diploid strain arg-1xpab-2 homozygous for the arg-7 locus. No difference was found in growth rate and in the Michaelis constant values for ASL. The specific activity of the enzyme produced in the heterozygotes was about 50 percent of the activity found in haploid or diploid wild-type. The heat sensitivity of ASL was also investigated in the different strains: two (containing the complementing mutations arg-7-5 and arg-7-7) of the four heterozygotes produce ASL varieties different from the wild-type enzyme as far as the thermolability is concerned.These results suggest that hybrid ASL can be formed by interaction between the products of wild-type and mutant genes. A clear dominance of the wild-type allele is expected only when the mutant allele has no product of the gene: this could be the case for arg-7-1 and arg-7-6.     

An analysis of the embryonic defects in Punch mutants of Drosophila melanogaster

Punch (Pu), a complex genetic locus, encodes GTP cyclohydrolase, the first enzyme in the pteridine biosynthetic pathway. In the larval and adult stages of the Drosophila life cycle, the function of the locus can be monitored by enzyme assays. Although enzyme activity cannot be detected prior to larval stages, the locus must also have earlier functions since most homozygous Pu mutants die during embryogenesis. In order to assess the role of the locus during this stage of development, morphological examinations of embryos from different classes of Pu mutants were performed. An exact correspondence has been found between genetic and morphological classes of Pu mutations. The locus is required during two periods of embryogenesis. These requirements are genetically separable as shown by mutants with defects specific to each period. An early function utilizes both maternal and zygotic components. Mutants defective for these components have abnormal segment patterns. Late in embryogenesis, a Pu product is necessary for the proper pigmentation of larval cuticle and proper orientation and differentiation of other larval structures, particularly in the head region. A cold-sensitive period corresponds to this later function as determined by temperature-shift experiments. Some of the phenotypes observed correspond to known physiological roles of pteridines; others are unexpected and unexplained.     

Cytogenetic analysis of two ecdysone-regulated puffs 62C and 62E in Drosophila melanogaster

Genetic analysis has been performed to reveal vital genes around two puffs, a late 62C puff and an early-late 62E puff. Their roles in hormonal regulatory mechanisms have been estimated. A locus represented by four lethal mutations has been found in the vicinity of the 62E puff. The mutants display disturbed puffing, which suggests the involvement of this locus in hormonal regulatory mechanisms. In the 62C puff region. 26 mutations have been found that proved to be allelic to mutations in the D-Titin gene. The giant D-Titin gene is essential for the sarcomeric organization of striated muscles. According to the results of in situ hybridization with polytene chromosomes, the D-Titin gene occupies the entire 62C puff. The phenotypic characteristics of the novel mutants suggest that this protein is polyfunctional, and its role is not restricted to processes in the muscular tissue. It may also be involved in the morphogenesis of leg imaginal disks, and it is necessary for condensation and separation of sister chromatids during mitosis. Mutations in the ecdysone-induced BR-C and E74 genes cause disturbances similar to those found in this study. In addition, mutations of these genes can affect the D-Titin gene activity, which suggests that the three genes are involved in similar morphogenetic and myogenetic processes.     

Genetic Analysis of Mutants of SACCHAROMYCES CEREVISIAE Resistant to the Herbicide Sulfometuron Methyl

Sulfometuron methyl (SM), a potent new sulfonylurea herbicide, inhibits growth of the yeast Saccharomyces cerevisiae on minimal media. Sixty-six spontaneous mutants resistant to SM were isolated. All of the resistance mutations segregate 2:2 in tetrads; 51 of the mutations are dominant, five are semidominant and ten are recessive. The mutations occur in three linkage groups, designated SMR1, smr2 and smr3. Several lines of evidence demonstrate that the SMR1 mutations (47 dominant and four semidominant) are alleles of ILV2 which encodes acetolactate synthase (ALS), the target of SM. First, SMR1 mutations result in the production of ALS enzyme activity with increased resistance to SM. Second, molecular cloning of the ILV2 gene permitted the isolation of mutations in the cloned gene which result in the production of SM-resistant ALS. Finally, SMR1 mutations map at the ILV2 locus. The smr2 mutations (four recessive, two dominant and one semidominant) map at the pdr 1 (pleiotropic drug resistance) locus and show cross-resistance to other inhibitors, typical of mutations at this locus. The smr3 mutations (six recessive and two dominant) define a new gene which maps approximately midway between ADE2 and HIS3 on the right arm of chromosome XV.     

Genes involved in meso-diaminopimelate synthesis in Bacillus subtilis: identification of the gene encoding aspartokinase I

Thermosensitive mutants of Bacillus subtilis deficient in peptidoglycan synthesis were screened for mutations in the meso-diaminopimelate (LD-A2pm) metabolic pathway. Mutations in two out of five relevant linkage groups, lssB and lssD, were shown to induce, at the restrictive temperature, a deficiency in LD-A2pm synthesis and accumulation of UDP-MurNAc-dipeptide. Group lssB is heterogeneous; it encompasses mutations that confer deficiency in the deacylation of N-acetyl-LL-A2pm and accumulation of this precursor. Accordingly, these mutations are assigned to the previously identified locus dapE. Mutations in linkage group lssD entail a thermosensitive aspartokinase 1. Therefore, they are most likely to affect the structural gene of this enzyme, which we propose to designate dapG. Mutation pyc-1476, previously reported to affect the pyruvate carboxylase, was shown to confer a deficiency in aspartokinase 1, not in the carboxylase, and to belong to the dapG locus, dapG is closely linked to spoVF, the putative gene of dipicolinate synthase. In conclusion, mutations affecting only two out of eight steps known to be involved in LD-A2pm synthesis were uncovered in a large collection of thermosensitive mutants obtained by indirect selection. We propose that this surprisingly restricted distribution of the thermosensitive dap mutations isolated so far is due to the existence, in each step of the pathway, of isoenzymes encoded by separate genes. The biological role of different aspartokinases was investigated with mutants deficient in dapE and dapG genes. Growth characteristics of these mutants in the presence of various combinations of aspartate family amino acids allow a reassessment of a metabolic channel hypothesis, i.e. the proposed existence of multienzyme complexes, each specific for a given end product.     

Estimation of mutation induction rates in AT-rich sequences using a genome scanning approach after X irradiation of mouse spermatogonia

We have previously used NotI as the marker enzyme (recognizing GCGGCCGC) in a genome scanning approach for detection of mutations induced in mouse spermatogonia and estimated the mutation induction rate as about 0.7 x 10(-5) per locus per Gy. To see whether different parts of the genome have different sensitivities for mutation induction, we used AflII (recognizing CTTAAG) as the marker enzyme in the present study. After the screening of 1,120 spots in each mouse offspring, we found five mutations among 92,655 spots from the unirradiated paternal genome, five mutations among 218,411 spots from the unirradiated maternal genome, and 13 mutations among 92,789 spots from 5 Gy-exposed paternal genome. Among the 23 mutations, 11 involved mouse satellite DNA sequences (AT-rich), and the remaining 12 mutations also involved AT-rich but non-satellite sequences. Both types of sequences were found as multiple, similar-sequence blocks in the genome. Counting each member of cluster mutations separately and excluding results on one hypermutable spot, the spontaneous mutation rates were estimated as 3.2 (+/- 1.9) x 10(-5) and 2.3 (+/- 1.0) x 10(-5) per locus per generation in the male and female genomes, respectively, and the mutation induction rate as 1.1 (+/- 1.2) x 10(-5) per locus per Gy. The induction rate would be reduced to 0.9 x 10(-5) per locus per Gy if satellite sequence mutations were excluded from this analysis. The results indicate that mutation induction rates do not largely differ between GC-rich and AT-rich regions: 1 x 10(-5) per locus per Gy or less, which is close to 1.08 x 10(-5) per locus per Gy, the current estimate for the mean mutation induction rate in mice.