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PubMedDate not supplied

Action of physiologically active materials in human semen during aging.

We examined the activities of some physiologically active substances (enzymes) in human seminal plasma from subjects in their 20s, 30s, and 40s. The average volume of semen per ejaculation in this investigation did not differ significantly depending on the age of the subject. Two age dependent patterns of decrease of substances in semen were observed, and the substances including tissue kallikrein and prostate specific antigen (PSA) basic arginine amidase in human seminal plasma showing the first pattern (a significant decrease in the 40s as compared to the 30s) might be initially secreted from the prostate gland, and whereas the glands secreting the other group of substances including active form coagulation factor X (FXa) and plasminogen are not now known. The levels of these substances in semen decrease in the subjects in their 30s. The coagulation and liquefaction times of human semen from older subjects were both prolonged with those of semen from younger subjects, and that such alteractions ultimately cause the age dependent declines of the motility of sperm and the ability of fertility.

Adult↗

PubMed2004

Differential expression of mycothiol pathway genes: are they affected by antituberculosis drugs?

Mycothiol (MSH) is the major cellular thiol in Mycobacterium tuberculosis (M.tb). We hypothesize that the mycothiol-dependent detoxification pathway may serve an important role during oxygen stress management in M. tuberculosis, derived from normal aerobic metabolism, the macrophage environment and through the action of anti-tubercular antibiotics, such as Isoniazid (INH). Total mRNA and DNA were isolated from M. bovis BCG at different stages of growth in 7H9 mycobacterial medium. Three genes involved in mycothiol metabolism and encoding the enzymes mycothiol S-conjugate amidase (Mca, Rv1082), NADPH dependent mycothiol reductase (mtr, Rv2855), and N-Acetyl-1-D-myo-Inosityl-2-Amino-2-Deoxy-alpha-D-Glucopyranoside Deacetylase (GlcNAc-Ins deacetylase, Rv1170 or mshB) were investigated for genomic rearrangements and expression. The results show that the genomic domains of the genes remain conserved in evolutionary diverse and unrelated M. tuberculosis isolates. The genes encoding enzymes implicated in mycothiol reduction, mtr (Rv2855) and the mycothiol-dependant detoxification of electrophilic agents, Mca (Rv1082), are shown to be actively transcribed during logarithmic M. bovis BCG growth. The gene encoding GlcNAc-Ins deacetylase (the rate limiting mycothiol biosynthesis step) shows induction in the presence of INH. Antisense oligonucleotides to both GlcNAc-Ins deacetylase (Rv1170) and mtr (Rv2855) mRNA affect mycobacterial growth. In conclusion the results presented here suggest that these enzymes are sensitive to free radical generating antituberculosis drugs and may be useful targets for new drug development.

Acetylglucosamine↗

PubMed1997

Identification of an "alcohol dehydrogenase-activating" protease in grass carp hepatopancreas as a chymotrypsin.

Previous investigation [Tsui et al. (1996) Biochim. Biophys. Acta 1269: 41-46] showed that two active forms of alcohol dehydrogenase can be purified from grass carp. The use of a protease inhibitor and the results of SDS-PAGE analysis of the enzymes suggest that one form (ADH-C) is a proteolytic product of the other (ADH-I). In this study, the protease responsible for the cleavage was purified. The cleavage enzyme had a subunit molecular weight of 28 kDa. An inhibitor study identified it as a serine protease. It exhibited a strong chymotrypsin activity in both esterase and amidase assays with a pH optimum in the range 7.5-8.5. The purified chymotrypsin also cleaved the intact grass carp ADH-I into the two-fragment ADH-C, with an accompanying increase in enzyme activity. A similar effect was not found using horse liver alcohol dehydrogenase.

Alcohol Dehydrogenase↗

PubMed2004

Tumor-activated prodrugs--a new approach to cancer therapy.

Systemic cytotoxic (antiproliferative) anticancer drugs rely primarily for their therapeutic effect on cytokinetic differences between cancer and normal cells. One approach aimed at improving the selectivity of tumor cell killing by such compounds is the use of less toxic prodrug forms that can be selectively activated in tumor tissue (tumor-activated prodrugs; TAP). There are several mechanisms potentially exploitable for the selective activation of TAP. Some utilize unique aspects of tumor physiology such as selective enzyme expression or hypoxia. Others are based on tumor-specific delivery techniques, including activation of prodrugs by exogenous enzymes delivered to tumor cells via monoclonal antibodies (ADEPT) or generated in tumor cells from DNA constructs containing the corresponding gene (GDEPT). Whichever activating mechanism is used, only a small proportion of the tumor cells are likely to be competent to activate the prodrug. Therefore, TAP need to fully exploit these "activator" cells by being capable of killing activation-incompetent cells as well via a "bystander effect." A wide variety of chemistries have been explored for the selective activation of TAP. Examples are given of the most important-the reduction of quinones, N-oxides, and nitroaromatics by endogenous enzymes or radiation; the cleavage of amides by endogenous peptidases; and hydrolytic metabolism by a variety of exogenous enzymes, including phosphatases, kinases, amidases, and glycosidases.

Antibodies, Neoplasm↗

PubMed1995

beta-Lactamase induction in gram-negative bacteria is intimately linked to peptidoglycan recycling.

A number of Gram-negative organisms normally express a chromosomally mediated class C beta-lactamase that is inducible by beta-lactam antibiotics. Data have recently emerged suggesting a close link between beta-lactamase induction and the recycling of released muramyl peptides from the bacterial peptidoglycan. Thus the AmpG transporter is responsible for the uptake into the cell of GlcNAc-anhMurNAc-tripeptide. A mutant unable to express AmpG is therefore unable to recycle the cell wall and is at the same time not possible to induce by a beta-lactam. Once inside the cytosol the above muramyl peptide and its derivative anhMurNAc-tripeptide is degraded by the cytosolic AmpD amidase that specifically releases the tripeptide from cytosolic muramyl peptides brought into the cell via AmpG. Mutants unable to produce AmpD are blocked in a cytosolic step for cell wall recycling and accumulate large amounts of cytosolic anhMurNAc-tripeptide. It is believed that cytosolic muramyl peptides can act as ligands for the beta-lactamase regulator AmpR to activate expression of beta-lactamase. AmpD mutants, therefore, constitutively overproduce the chromosomal beta-lactamase and are beta-lactam resistant. In wild-type strains beta-lactams that result in an increased cell wall breakdown will cause an increase in the cytosol of muramyl peptides leading to beta-lactamase induction. Mutants affected in the ampD gene arise readily during treatment with third-generation cephalosporins. Since these mutants lack a functional cell wall recycling system they may be at a disadvantage in the absence of selection. However, since muramyl peptides may act as cytotoxins, especially for respiratory epithelial cells, ampD mutants due to their large accumulation of anhMurNAc-tripeptide may be altered in their pathogenic properties as compared to wild-type cells possessing a normal cell wall recycling system.

Enzyme Induction↗

PubMed1980

Ampicillin resistance in Hemophilus parainfluenzae.

Ampicillin resistance among strains of Hemophilus is usually due to production of beta-lactamase. This paper reports the isolation of a strain of H. parainfluenzae resistant to ampicillin with no detectable beta-lactamase or amidase activity. The organism, isolated from the blood of a patient who had aortic valve endocarditis, gave a zone diameter consistent with ampicillin sensitivity when tested by disc diffusion in Mueller-Hinton agar supplemented with 1% IsoVitaleX and 1% hemoglobin. Broth dilution testing in Levinthal medium, however, revealed the following minimal inhibitory cencentrations: ampicillin, 32 micrograms/ml; penicillin, 256 micrograms/ml; methicillin, 128 micrograms/ml; carbenicillin, 128 micrograms/ml; and cephalothin and chloramphenicol, 1.0 micrograms/ml. The results of acidimetric, iodometric, and chromogenic cephalosporin methods for detection of beta-lactamase were negative. Beta-lactamase activity could not be demonstrated in cell sonicates or induced by growth of the cells in antibiotic-containing medium. In addition, no extracellular degradation of either ampicillin or penicillin could be demonstrated.

Adult↗

PubMed1988

The essential role of microsomal deacetylase activity in the metabolic activation, DNA-(deoxyguanosin-8-yl)-2-aminofluorene adduct formation and initiation of liver tumors by N-hydroxy-2-acetylaminofluorene in the livers of infant male B6C3F1 mice.

Deacetylation of N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) to N-hydroxy-2-aminofluorene (N-hydroxy-AF) has been proposed as one of the critical metabolic steps in the formation of hepatic DNA adducts and the initiation of liver tumors in 12-day-old male B6C3F1 mice. In this study, the importance of the microsomal deacetylase activity for N-hydroxy-AAF in the initiation of hepatocarcinogenesis in these mice was demonstrated by using a carboxylesterase and amidase inhibitor, bis(p-nitrophenyl)phosphate (BNPP), that is much less toxic in vivo than is paraoxon. Pre-incubation of liver microsomes from 12-day-old male B6C3F1 mice with 10(-3) M BNPP reduced the deacetylase activity by 80% while paraoxon inhibited the deacetylase activity completely at a concentration of 10(-4) M. Pretreatment of 12-day-old male B6C3F1 mice with 4 X 75 micrograms doses of BNPP/g body weight before the administration of N-hydroxy-AAF reduced the hepatic N-(dGuo-8-yl)-AF adduct levels to 1.09 and 0.68 pmol/mg DNA compared with 2.87 and 1.64 pmol/mg DNA for mice treated once with 0.06 or 0.03 mumol of N-hydroxy-AAF/g body weight respectively. However, BNPP pretreatments did not affect the levels of the acetylated DNA adducts, N-(dGuo-8-yl)-AAF and 3-(dGuo-N2-yl)-AAF, formed by these doses of N-hydroxy-AAF. The initiation of liver tumors by N-hydroxy-AAF was also inhibited by BNPP pretreatment. Thus, for mice that received single doses of 0.12, 0.06 and 0.03 mumol of N-hydroxy-AAF/g body weight, the multiplicities of liver tumors at 10 months were reduced by BNPP pretreatments to 5.6, 1.0 and 0.3 compared with multiplicities of 11.8, 4.8 and 1.7 without pretreatment respectively. On the other hand, BNPP pretreatments had no significant inhibitory effects on the levels of the hepatic DNA-N-(dGuo-8-yl)-AF adduct or on the liver tumor multiplicities induced by comparable doses of N-hydroxy-AF. It is concluded that deacetylation of N-hydroxy-AAF to N-hydroxy-AF is essential for the metabolic activation, DNA-N-(dGuo-8-yl)-AF adduct formation and liver tumor initiation in infant male B6C3F1 mice by N-hydroxy-AAF.

2-Acetylaminofluorene↗

PubMed1989

Enzymes in normally perfused and ischaemic dog hearts which release a substance with kinin like activity.

The presence of amidases cleaving the tripeptide VAL.LEU.ARG.pNA, and liberating from human plasma kininogen substance(s) able to contract uterine smooth muscle and to lower blood pressure (uterus contracting and hypotensive activity), has been demonstrated in vascular and muscle tissues from normally perfused and ischaemic areas of dog hearts. Studies were carried out on blood free preparations of coronary arteries and veins and normally perfused and ischaemic ventricle. All the tissues were found to contain both acid optimum (pH 6) and alkaline optimum (pH greater than 9) enzymes forming uterus contracting substance (UCS, bioassayed on isolated uterus of rats in oestrus), the highest levels of both activities being found in arterial tissues and the least in ventricle. Enzyme levels in ischaemic or normally perfused ventricle did not differ significantly. Gel filtration (Sephacryl, S-300) of coronary artery extracts gave one peak each of acid optimum enzyme with a molecular weight of 38,300 +/- 800 daltons and alkaline optimum enzyme with a molecular weight of 92,100 +/- 4000 daltons. Both acid and alkaline enzyme fractions cleaved the tripeptide substrate with pH optima identical to those for UCS formation. The acid optimum activity, both of UCS formation and tripeptide cleavage, was inhibited by pepstatin but not by aprotinin or soybean trypsin inhibitor (SBTI). The alkaline optimum activity was inhibited by aprotinin and SBTI but not pepstatin. Both acid and alkaline optimum enzymes released a hypotensive agent from a plasma protein substrate. The molecular weight and response to inhibitors of the acid optimum enzyme were similar to a cathepsin, and those of the alkaline optimum enzyme were similar to plasma kallikrein.

Animals↗

PubMed1999

Crystal structure and induction mechanism of AmiC-AmiR: a ligand-regulated transcription antitermination complex.

Inducible expression of the aliphatic amidase operon in Pseudomonas aeruginosa is controlled by an antitermination mechanism which allows production of the full-length transcript only in the presence of small-molecule inducers, such as acetamide. Ligand-regulated antitermination is provided by AmiC, the ligand-sensitive negative regulator, and AmiR, the RNA-binding positive regulator. Under non-inducing or repressing growth conditions, AmiC and AmiR form a complex in which the activity of AmiR is silenced. The crystal structure of the AmiC-AmiR complex identifies AmiR as a new and highly unusual member of the response-regulator family of bacterial signal transduction proteins, regulated by sequestration rather than phosphorylation. Comparison with the structure of free AmiC reveals the subtle mechanism of ligand-induced release of AmiR.

Amino Acid Sequence↗

PubMed2002

Structure of malonamidase E2 reveals a novel Ser-cisSer-Lys catalytic triad in a new serine hydrolase fold that is prevalent in nature.

A large group of hydrolytic enzymes, which contain a conserved stretch of approximately 130 amino acids designated the amidase signature (AS) sequence, constitutes a super family that is distinct from any other known hydrolase family. AS family enzymes are widespread in nature, ranging from bacteria to humans, and exhibit a variety of biological functions. Here we report the first structure of an AS family enzyme provided by the crystal structure of malonamidase E2 from Bradyrhizobium japonicum. The structure, representing a new protein fold, reveals a previously unidentified Ser-cisSer-Lys catalytic machinery that is absolutely conserved throughout the family. This family of enzymes appears to be evolutionarily distinct but has diverged to acquire a wide spectrum of individual substrate specificities, while maintaining a core structure that supports the catalytic function of the unique triad. Based of the structures of the enzyme in two different inhibited states, an unusual action mechanism of the triad is proposed that accounts for the role of the cis conformation in the triad.

Amidohydrolases↗

PubMed2001

The formamidase gene of Aspergillus nidulans: regulation by nitrogen metabolite repression and transcriptional interference by an overlapping upstream gene.

The ability to utilize formamide as a sole nitrogen source has been found in numerous fungi. We have cloned the fmdS gene encoding a formamidase from Aspergillus nidulans and found that it belongs to a highly conserved family of proteins separate from the major amidase families. The expression of fmdS is primarily regulated via AreA-mediated nitrogen metabolite repression and does not require the addition of exogenous inducer. Consistent with this, deletion analysis of the 5' region of fmdS has confirmed the presence of multiple AreA-binding sites containing a characteristic core GATA sequence. Under carbon starvation conditions the response to nitrogen starvation is eliminated, indicating that the lack of a carbon source may result in inactivation of AreA. Sequence analysis and isolation of cDNAs show that a gene of unknown function lies directly 5' of fmdS with its transcript overlapping the fmdS coding region. Disruption of the 5' gene and analysis of the effects of overexpression of this gene on fmdS expression has shown that expression of this upstream gene interferes with fmdS transcription, resulting in a strong dependence on AreA activation for expression. Therefore the relative position of these two genes is essential for normal regulation of fmdS.

Amidohydrolases↗

PubMed1992

Structural analysis of the N-linked glycan chains from a stylar glycoprotein associated with expression of self-incompatibility in Nicotiana alata.

Self-incompatibility in flowering plants of the family Solanaceae is mediated by the product of the S-allele. The allelic products of the S-gene in the female sexual tissues of the pistil are glycoproteins in the mol. wt range 28-32 kDa. These S-glycoproteins have been isolated from styles of Nicotiana alata, homozygous for the S1- and S2-alleles. Earlier studies have indicated that the single potential N-glycosylation site on the S1-glycoprotein bears a glycan chain, whereas of the four potential N-glycosylation sites on the S2-glycoprotein, three are glycosylated. This paper describes the purification and characterization of the N-linked glycan chains from these two glycoproteins. Oligosaccharides were cleaved off the glycoproteins using peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (N-glycanase F) and separated by anion-exchange HPLC. Four types of hybrid structure were defined by chemical techniques, fast atom bombardment-mass spectrometry (FAB-MS) and 1H-NMR. Although the relative amounts differed, all four structures were found on both the S1- and S2-glycoproteins, and are heterogeneous at some N-glycosylation sites. No O-linked glycans were detected on the S2-glycoprotein. These results are discussed in relation to the potential of the structural diversity residing in this array of glycoforms to play a rôle in allelic specificity.

Alleles↗

PubMed1993

2-Iminothiolane: a reagent for the introduction of sulphydryl groups into oligosaccharides derived from asparagine-linked glycans.

A facile method for introducing reactive sulphydryl groups into oligosaccharides was developed. 1-Amino-oligosaccharides generated from asparagine-linked glycans by peptide-N4(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase F) digestion were monitored by high-performance anion-exchange chromatography with pulsed amperometric detection and derivatized under optimal conditions with 2-iminothiolane-HCl. The resulting mercapto-butyramido oligosaccharides, which were obtained in high yield, were alkylated with a fluorescent reagent and used to selectively assay for endoglycosidases that hydrolyse di-N-acetylchitobiose linkages.

Asparagine↗

PubMed1993

Glycosylation pattern and processing of envelope gene products encoded by glycosylation mutants of Friend spleen focus-forming virus.

The protein encoded by the envelope gene of Friend spleen focus-forming virus is responsible for the acute leukaemogenicity of this virus. In order to correlate glycosylation and intracellular processing of this protein with viral pathogenicity, envelope gene products of pathogenic and apathogenic glycosylation mutants were expressed in Rat-1 cells and metabolically labelled with [6-3H]glucosamine. Following immunoprecipitation, primary and secondary gene products (gp55, gp65) were separated by preparative polyacrylamide gel electrophoresis. Oligosaccharides were released from tryptic glycopeptides by treatment with endo-beta-N-acetylglucosaminidase H (gp55), peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (gp65) or by reductive beta-elimination. Resulting glycans were characterized by co-chromatography with authentic oligosaccharide standards using different HPLC systems and digestion with exoglycosidases. The results revealed that the primary envelope gene products of pathogenic glycosylation mutants were, in part, further processed in Rat-1 cells similar to wild-type glycoprotein, resulting in polypeptides carrying complex-type N-glycans as well as partially sialylated O-linked oligosaccharides. In contrast, corresponding glycoproteins encoded by apathogenic mutants were found to remain at the level of the primary translation product exclusively comprising high-mannose-type N-glycans. Hence, intracellular maturation of the envelope gene products in this model cell line seems to correlate with the in vivo pathogenicity of the glycosylation mutants studied.

Animals↗

PubMed1994

Oligosaccharide profiles of HIV-2 external envelope glycoprotein: dependence on host cells and virus isolates.

The glycosylation pattern of the external envelope glycoprotein of human immunodeficiency virus type 2 (HIV-2) was studied in dependence on host cells and virus isolates. Strains HIV-2ALT, HIV-2ROD and HIV-2D194, differing in their biological properties and in the amino acid sequences of their env genes, were propagated in MOLT4, HUT78 and U937 cells, in human peripheral blood lymphocytes and monocytes/macrophages in the presence of [6-3H]glucosamine. Radiolabelled viral glycoproteins were isolated from the cell-free supernatants and digested with trypsin. Glycans were sequentially liberated by endo-beta-N-acetylglucosaminidase H and peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F, and fractionated according to charge and size. Comparison of the oligosaccharide profiles revealed that the envelope glycoproteins of different virus isolates, propagated in the same host cells, yielded very similar glycan patterns, whereas cultivation of an isolate in different host cells resulted in markedly divergent oligosaccharide maps. Variations concerned the proportion of high-mannose-, hybrid- and complex-type substituents, as well as the state of charge and structural parameters of the complex-type species. As a characteristic feature, complex-type glycans of macrophage-derived viral glycoprotein were almost exclusively substituted by lactosamine repeats. Hence, glycosylation of the HIV-2 external envelope glycoprotein seems to be primarily governed by host cell-specific factors rather than by the amino acid sequence of the corresponding polypeptide backbone.

Carbohydrate Sequence↗

PubMed1997

Analysis of site-specific N-glycosylation of recombinant Desmodus rotundus salivary plasminogen activator rDSPA alpha 1 expressed in Chinese hamster ovary cells.

The recombinant plasminogen activator (rDSPA alpha 1) from the vampire bat Desmodus rotundus is a promising new thrombolytic agent that exhibits a superior pharmacological profile if compared to tissue-type plasminogen activator (t-PA) or streptokinase. In the present study the structures of the carbohydrate moieties at the two N-glycosylation sites (Asn-117, Asn-362) of rDSPA alpha 1 expressed in Chinese hamster ovary cells were determined. N-Linked glycans were enzymatically released from isolated tryptic glycopeptides by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F digestion and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by analysis of carbohydrate composition and linkage, by mass spectrometry, and by sequence analysis in which the fluorescently labeled glycans were cleaved with an array of specific exoglycosidases. More than 30 different oligosaccharides were identified. The results revealed that Asn-117 carried a mixture of one high-mannose structure (17% of site-specific glycosylation), three hybrid glycans (26%) and predominantly biantennary complex N-glycans (54%). Glycosylation site Asn-362 was found to comprise complex glycans with biantennary (50%), 2,4- and 2,6-branched triantennary (21%, 11%), and tetraantennary structures (10%), which were fucosylated at the innermost residue of N-acetylglucosamine. Mainly neutral and monosialylated glycans, and smaller quantities of disialylated glycans, were detected at both glycosylation sites. Sialic acid was alpha 2-3 linked to galactose exclusively. As shown in this study the N-glycans attached to Asn-117 of rDSPA alpha 1 are more processed during biosynthesis than the high-mannose structures linked to Asn-117 of t-PA, to which the polypeptide backbone of rDSPA alpha 1 is structurally closely related.

Amino Acid Sequence↗

PubMed2003

N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits.

Human C1 inhibitor (hC1INH) is a therapeutic N, O-glycoprotein with a growing number of clinical applications, but the current natural supplies are not likely to meet the clinical demands. Therefore, recombinant approaches are of interest, whereby specific attention has to be paid to the generated glycosylation patterns. Here, the N,O-glycoprotein was expressed in the mammary gland of transgenic rabbits and subjected to glycan analysis. After release of the N-glycans of recombinant-rabbit human C1 inhibitor (rhC1INH) by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the O-glycoprotein by centrifugal filtration, then fractionated by a combination of anion-exchange, normal-phase, and high-pH anion-exchange liquid chromatography. The O-glycans, released from the O-glycoprotein by alkaline borohydride treatment, were fractionated by anion-exchange high-performance liquid chromatography (HPLC). The structures of individual components were analysed by 500 MHz 1H NMR spectroscopy, in most cases combined with MALDI-TOF MS. In contrast to the structural data reported for native serum hC1INH, rhC1INH contained a broad array of different N-glycans, made up of oligomannose-, hybrid-, and complex-type structures. In the case of complex-type N-glycans (partially) (alpha2-6)-sialylated (N-acetylneuraminic acid only), mono- and diantennary chains were found; part of the diantennary structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the lower or upper antenna (Lewis x). The manno-oligosaccharide pattern of part of the hybrid- and oligomannose-type structures indicates that besides the usual N-glycan processing route, also the alternative endo-mannosidase pathway is followed. The small core 1-type O-glycans showed the usual (alpha2-3)- and (alpha2-6)-sialylation pattern of O-glycoproteins of nonmucinous origin.

Animals↗

PubMed1995

Deletion of the C-terminal end of aspartylglucosaminidase resulting in a lysosomal accumulation disease: evidence for a unique genomic rearrangement.

Aspartylglucosaminuria (AGU) is an inborn error of glycoprotein catabolism and represents the only known human deficiency of an amidase, aspartylglucosaminidase (AGA, EC 3.5.1.26). We report here a detailed characterization of a unique 2 kb deletion of the AGA gene in a North American AGU patient. To facilitate the characterization of the deletion, genomic lamda clones spanning the 3' flanking region of human AGA were isolated and sequenced. The breakpoint of the deletion was determined from the patient's DNA by sequencing the genomic region containing the novel junction. The rearrangement involved a nonhomologous recombination with only 2 bp of homology at the deletion breakpoint. The deletion's 5' breakpoint was located in the last intron of AGA, thus abolishing the normal C-terminal exon. This is in contrast to our previous findings indicating that the deletion in the AGA gene would contain only the complete 3' untranslated region and leave the coding region intact (1). The unique feature of this deletion is a triplication of 19 thymidine nucleotides of an inverted Alu repeat, which is located at the deletion 3' breakpoint. The analysis of the patient's AGA cDNA revealed an open reading frame containing a novel C-terminal exon, coding for a 64 amino acid sequence, which has no homology to the normal exon 9 of AGA. This new exon has a functional splice acceptor site at its 5' end, a stop codon, and a polyadenylation signal at the 3' end. Expression of the mutant AGA cDNA in COS cells showed that mutant mRNA is synthesized in equal amounts compared with normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗