AAT Gene Research Articles

Haplotypes of the alpha-1 antitrypsin gene in healthy controls and Z deficiency patients

Alpha-1 antitrypsin (AAT; HUGO symbol, SERPINA1) is one of the major serine protease inhibitors (serpins) in human plasma. Deficiency of AAT is a recognised risk factor for chronic obstructive pulmonary disease (COPD), attributed to uninhibited neutrophil elastase released into the lung tissue during inflammatory states. In this study we used sequencing to screen the exonic regions, 5' and 3' flanking sequence of the AAT gene in order to generate a high density map of single nucleotide polymorphisms (SNPs). 16 SNPs were identified throughout AAT. Haplotypes based on SNPs with a minor allele frequency of > or =5% were estimated using genotypic information from 225 healthy control individuals and 41 AAT deficient Pi-ZZ individuals. AAT shows a large amount of variation in the control population, with 17 haplotypes accounting for 88% of the observed variation. The haplotype distribution of the common deficiency Pi-Z variant of AAT was significantly different when compared to the normal variants. In addition to the haplotype information, we present evidence for a functional effect of a SNP in intron 1.

Absence of Alpha-1 Antitrypsin Deficiency Alleles ( S and Z ) in Japanese and Korean Patients With Aneurysmal Subarachnoid Hemorrhage

A possible association has been proposed for the formation of intracranial aneurysm (IA) and deficiency alleles (S and Z) of the alpha1-antitrypsin (AAT) gene. We extensively screened this gene in Japanese and Korean patients with aneurysmal subarachnoid hemorrhage. Seven allelic variants, including S and Z alleles, were genotyped by direct sequencing of genomic DNA obtained from 195 and 189 ruptured IA patients and 195 and 94 controls in Japanese and Koreans, respectively. The haplotype in phase-unknown samples was constructed with the expectation-maximization method. Differences in allelic frequencies between patients and controls were evaluated by Fisher exact test. No significant differences in allelic frequencies were observed at all 7 variants between ruptured IA patients and controls. We could not detect the S and Z alleles of the AAT gene in Japanese and Korean populations. AAT deficiency may not be a common genetic risk factor for aneurysmal subarachnoid hemorrhage in Japanese and Koreans.

"The lion, the witch and the wardrobe": impact on sibs of individuals with AAT deficiency.

Alpha(1)-antitrypsin (AAT) deficiency is a genetic disorder that may cause serious pulmonary or liver impairment in children or adults. Although genetic sequencing of the AAT gene has only been available for 20 years, analysis of the amount and electrophoretic mobility of the AAT protein has allowed clinical phenotyping for more than 40 years. There have been no studies assessing the psychological impact of having a sib affected by AAT deficiency. Twenty-five participants drawn from the Alpha-1 Research Registry completed a questionnaire and semi-structured interview. Respondents were supportive of testing prior to adulthood for AAT status; 18 thought it was a good idea to test a child, three did not know, and four said children should not be tested, primarily citing insurance concerns. Of those 18 who stated it was a good idea, 14 would test at birth. Knowledge of genetics of AAT deficiency was limited; only 44% of respondents understood the inheritance pattern. We recommend: (1) parents and sibs need help in mourning the loss of children with AAT deficiency; young sibs are at risk for trauma and long-term developmental problems. (2) Teams evaluating donors for liver transplantation should be aggressive in ruling out AAT deficiency prior to invasive testing. (3) Testing should be offered to individuals with a family history of AAT deficiency to obtain the health benefits of lifestyle modification and limit the burden of disease discovery in symptomatic relatives. (4) Awareness of liver disease from AAT deficiency should be increased.

Aroma Production in Climacteric Fruit: A Comparison of Tomato, Apple, and Banana

Volatile aroma compounds produced by apple, banana, and tomato are produced throughout development, however, those associated with ripening and edible quality are dependent upon ethylene action. In apple and banana, characteristic aroma is, in large part, dependent upon the formation of volatile esters. In tomato, many of the characteristic aromas are dependent upon tissue disruption and result from aldehydes and alcohols following lipid degradation. For apple and banana, the enzyme alcohol acyl-CoA transferase (AAT, EC 2.3.1.84) is the enzyme responsible for the final reaction in the pathway for ester formation and catalyzes the union of an alcohol and the CoA derivative of fatty acids. In both tissues, AAT gene expression was detected prior to the onset of ester production. In apple, AAT expression was found to be closely tied with the onset of autocatalytic ethylene synthesis. In banana, ethylene synthesis peaked and began to decline well before ester synthesis began. However, the expression of AAT increased as ester production increased for both tissues. Tomato fruit, like apple and banana, produced characteristic aromas following the onset of the ethylene climacteric, suggesting changes in the activity of various components of the lipoxygenase pathway. In all three tissue types, there are continuous, significant shifts in the aroma profile as fruit ripen age, suggesting shifts in specific metabolic pathways associated with precursor synthesis or degradation.

101. Toxicology and Biodistribution Studies of a Recombinant Adeno-Associated Virus 2 (rAAV2) Alpha-1 Antitrypsin (AAT) Vector

A series of safety and biodistribution studies were undertaken to support the development of a muscle-based gene therapy system for AAT deficiency. AAT is the primary circulating serum proteinase inhibitor, and its deficiency can result in emphysema. The test article that was used in this toxicology study was rAAV serotype 2 with the CMV/Beta Actin promoter driving expression of the human AAT gene. There were 4 dosing arms in the primary toxicology study (PTS) with sacrifice time points being at 21 days and 90 days post vector administration. In the biodistribution study (BDS), there were 5 dosing arms with duplicate doses given intramuscularly and intravenously. The sacrifice time point for this study was at 60 days post vector administration. The PTS was performed in 48-C57/Bl6 mice (24M/24F) with the primary safety endpoint being the histopathologic examination of organs collected at necropsy. CBC and serum chemistry analyses were also performed. Gene transfer efficiency was evaluated from gDNA isolated from organs and whole blood by real-time PCR. The BDS was performed in 36-C57/Bl6 mice (18M/18F) to determine the potential for vector dissemination following test article administration IV and IM. In the PTS, no abnormalities were noted in the CBC and serum chemistry data among test article and control article study arms. Histopathology results were essentially normal with no vector related findings. Real Time PCR indicated that vector DNA was present in the peripheral blood 24 hours post administration, but decreased to near baseline levels at the time of sacrifice. Vector DNA was not detectable in gonad tissue. Real Time PCR results in the BDS also indicated vector DNA present in peripheral blood at 24 hours post vector administration. At sacrifice, IV injected mice vector levels were higher than IM injected mice. Vector DNA was found in skeletal muscle, lung, heart, liver, diaphragm, kidney and pancreas at the time of necropsy via IM administration at dose dependent levels. No vector DNA was detected in the skeletal muscle following IV administration, but substantial amounts were found in other organs. Gonads were negative for vector DNA with both IM and IV administration indicating that the risk of germline transmission with our gene therapy vector would be relatively low.

Are polymorphic markers within the alpha-1-antitrypsin gene associated with risk of human immunodeficiency virus disease?

We sought to determine whether variants of the human alpha-1-antitrypsin (AAT) gene, also known as "PI," or "SERPINA1," are associated with human immunodeficiency virus (HIV) infection in 2 African-based populations from HIV-pandemic sub-Saharan Africa. Eleven commonly occurring African-associated polymorphic markers in the coding and intronic regions of the AAT gene were analyzed via denaturing gradient gel electrophoresis. A significant association between HIV-1 infection and the presence of an allelic variant was observed in the case of the M2 and A332A haplotypes, thus presenting AAT as a potentially novel HIV-1 susceptibility locus.

Genetic diversity of the alpha-1-antitrypsin gene in Africans identified using a novel genotyping assay.

The highly polymorphic human alpha-1-antitrypsin (AAT) gene, more recently named SERPINA1, codes for the most abundant circulating plasma serine protease inhibitor, protease inhibitor 1 (PI). Most studies determining AAT haplotype frequencies have been restricted first by the limited accuracy of the phenotypic method used and secondly by the analysis of predominantly Caucasian populations. Limited studies have been performed on African-based populations. Here a new comprehensive assay for genotyping the entire coding region, including splice junctions, of the AAT gene was designed. This assay, based on denaturing gradient gel electrophoresis (DGGE), allows for the complete analysis of a single individual in two lanes on a gel. Application of the assay resulted in the identification of nine known AAT variants as well as 13 novel sequence variants, five of which are single nucleotide polymorphisms (SNPs), occurring exclusively in the African-based populations. This is the first comprehensive analysis of the genetic diversity of the AAT gene in a cohort from sub-Saharan Africa.

Oncostatin M induced alpha1-antitrypsin (AAT) gene expression in Hep G2 cells is mediated by a 3' enhancer.

alpha(1)-Antitrypsin (AAT) is the major serine proteinase inhibitor (SERPIN A1) in human plasma. Its target proteinase is neutrophil elastase and its main physiological function is protection of the lower respiratory tract from the destructive effects of neutrophil elastase during an inflammatory response. Circulating levels of AAT rise 2-3-fold during inflammation and the liver produces most of this increase. The cytokines oncostatin M (OSM) and interleukin-6 have been shown to be mainly responsible for this effect, which is mediated via the interaction of cytokine-inducible transcription factors with regulatory elements within the gene. In the present study, we report for the first time that OSM stimulation of hepatocyte AAT occurs via an interaction between the hepatocyte promoter and an OSM-responsive element at the 3'-end of the AAT gene. This effect is mediated by the transcription factor signal transducer and activator of transcription 3 ('STAT 3') binding to an OSM-responsive element (sequence TTCTCTTAA), and this site is distinct from, but close to, a previously reported interleukin-6-responsive element.

Lack of Association of Alpha-1 Antichymotrypsin Gene Polymorphism with PR3-ANCA and MPO-ANCA Associated Vasculitis

In patients with PR3-ANCA associated vasculitides the carrier frequency of f 1-antitrypsin (AAT) deficiency allele PI*Z is increased and linkage disequilibrium between polymorphic markers within a cluster of serine protease inhibitor (serpin) genes, including AAT gene, at chromosome 14q32.1 has been described. A 1 -antichymotrypsin (AACT), part of an extended serpin gene cluster, was discussed to contribute to PR3-ANCA associated vasculitis formation. To analyse, if an AACT gene polymorphism within the signal peptide sequence is associated with antineutrophil cytoplasm autoantibodies (ANCA) vasculitis allelic frequencies of AACT polymorphism were analysed in 128 control persons, 79 PR3-ANCA, and 30 MPO-ANCA patients. In MPO-ANCA patients also phenotyping of AAT was performed as well as frequency and linkage analysis of simple tandem repeat polymorphisms in the genes of cortisol-binding globulin, AAT, protein C inhibitor, and three extragenic markers (S48, S55, S51) of the gene cluster. Allelic frequencies of the AACT polymorphism did not differ between controls and vasculitis patients. In the MPO-ANCA group no patient expressed the Pi*Z defective allele of AAT and the allelic frequencies of polymorphic markers within the serpin gene cluster did not differ from those of the controls. Strong linkage disequilibrium was detected in MPO-ANCA patients neither. Therefore, we can not support the hypothesis that AACT polymorphism contributes to the pathogenesis of PR3-ANCA vasculitis. Nor is it probable that any factor, coded by the serpin gene cluster, contributes to MPO-ANCA vasculitis.

α1-Antitrypsin Deficiency as a Result of Compound Heterozygosity for the Z and MHeerlen Alleles

The specificity of many common rapid molecular techniques for only one or a few mutations may produce confusion when a rare mutation is present. We describe a patient in whom the MHeerlen allele of the AAT gene for the protease inhibitor α1-antitrypsin (AAT) was missed by widely used molecular genotyping (1)(2)(3)(4) and phenotyping methods for mutations in the AAT gene. The majority of these assays were developed to detect the S and Z mutations (Glu264→Val and Glu342→Lys, respectively), which are the most prevalent mutations in a Caucasian population (5). A 40-year-old woman with a serum AAT of 0.3 g/L (reference interval, 0.8–2.0 g/L; Beckman Array), suffering from dyspnea and a familial history of emphysema was genotyped as PIMZ. However, isoelectric focusing (IEF) analysis (Blood Transfusion Service, Amsterdam, The Netherlands) revealed only the Z isoform. Hence she was considered to be a ZZ phenotype, in agreement with her low serum AAT and her early emphysema. The only sibling/sister of the propositus was also affected by …

An alpha1-antitrypsin enhancer polymorphism is a genetic modifier of pulmonary outcome in cystic fibrosis.

Lung disease is the direct cause of death in over 90% of cystic fibrosis (CF) patients. Excess neutrophil elastase is an important determinant of pulmonary disease in CF. alpha1-antitrypsin (AAT), also known as alpha1-proteinase inhibitor (alpha1PI) is a major modulator of elastase activity. We investigated the hypothesis that an enhancer polymorphism in the AAT gene would contribute to pulmonary prognosis in CF. Respiratory function, chest X-ray scores, bacterial colonisation and infective exacerbation were assessed to evaluate pulmonary disease severity in the CF group. Sixteen patients were found to have the 1237A allele, and 108 the more frequent G allele. Contrary to expectation, the patients with the 1237A allele were found to have better indices of pulmonary disease progression than those without, as indicated by less change in X-ray score (1237A: 0.2+/-0.1; 1237G: 1.2+/-0.1; P = 0.002) and fewer infective exacerbations (1237A: 2.8+/-0.6; 1237G: 4.6+/-0.3; P = 0.03) over the preceding 2 years. Also, a higher proportion of the 1237A (25%) than the 1237G (6.5%) were not colonised by Pseudomonas Aeruginosa (P = 0.04). Prospective monitoring of infections for a further 2 years confirmed a lesser propensity to infection in patients with the 1237A allele. These trends were also observed in a tightly matched sub-set of CF genotypes of similar age and sex, thus confirming that these effects were independent of the CF genotype. These results indicate that this AAT enhancer polymorphism is associated with better pulmonary prognosis in CF. Though the number of CF patients with the polymorphism is small, and these data need to be confirmed in larger studies, they suggest that a cautious approach should perhaps be taken to treatment of CF patients with supplemental AAT.

Expression and purification of functional human alpha-1-Antitrypsin from cultured plant cells.

Human alpha-1-antitrypsin (AAT), the most abundant protease inhibitor found in the blood, was expressed in rice embryonic tissue suspension cell culture. This was accomplished by cloning the codon-optimized AAT gene into a vector containing the rice RAmy3D promoter and its signal sequence. The synthetic gene incorporates codons synonymous with those found in highly expressed rice genes. Approximately 1000 stable transformed calli were produced by particle bombardment mediated transformation and were screened for high AAT expression using a porcine elastase inhibitory activity assay. The band shift assay also confirmed that rice-derived AAT is functional regarding its binding capability to the elastase substrate. Time course studies were conducted to determine the optimum, postinduction expression levels from cell culture. AAT expression equivalent to 20% of the total secreted proteins was achieved, and a purification scheme was developed that yielded active AAT with purity greater than 95%. The potential applications of purified plant-derived AAT for treatments of various AAT-deficient diseases are discussed.

Purification and characterization of the alanine aminotransferase from the hyperthermophilic Archaeon pyrococcus furiosus and its role in alanine production.

Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6. 5 to 7.8 and at a temperature of over 95 degrees C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in the P. furiosus genome database. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. The k(cat)/K(m) values for alanine and pyruvate formation were 41 and 33 s(-1) mM(-1), respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aat gene. In addition, both the aat and gdh (encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdh genes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.

Regulation of the serine proteinase inhibitor (SERPIN) gene α1-antitrypsin: A paradigm for other SERPINs

α 1,-antitrypsin (AAT) is the archetypal member of the serine proteinase inhibitor (SERPIN) gene family. AAT is an acute-phase reactant and the plasma concentration increases three-to four-fold during the inflammatory response. In hepatocytes this increase is mediated primarily. by the cytokine interleukin-6 (IL-6) via the transcription factor NF-116. The AAT gene contains at least two enhancer elements, one at the 5′ end of the gene and the other at the 3′ end. Functional studies performed in mammalian hepatoma cells (Hep G2) using constructs containing these AAT enhancer regions linked to a reporter gene have demonstrated that the 5′ enhancer is dominant under basal conditions and that, following stimulation with IL-6, both enhancers are essential and the 3′ enhancer plays a major role. We have identified a mutation associated with lung disease which occurs in the 3′ AAT enhancer; the mutation occurs at a binding site for the ubiquitous transcription factor Oct-1. The functional significance of this mutation is a deficient IL-6 response. Using the AAT gene as a model, we describe the interactions which occur between transcription factors within the 3' enhancer and also those which take place between the 5′ and 3′ enhancers. These studies shed light on the molecular mechanism of the acute-phase response which could possibly be extended to other members of the SERPIN gene family.

Mutation in an α 1-antitrypsin enhancer results in an interleukin-6 deficient acute-phase response due to loss of cooperativity between transcription factors

We previously reported that a mutation in a 3′ enhancer region of the α 1-antitrypsin (AAT) gene is associated with chronic obstructive airways disease (COAD). During the acute-phase response the plasma concentration of AAT increases approximately 3-fold and this effect is mediated primarily by interleukin-6 (IL-6). We demonstrate, by transfection of Hep G2 cells, that the AAT gene contains at least two enhancers, one at the 5′ end of the gene which is dominant under basal conditions, and another at the 3′ end of the gene which exhibits weak basal activity. However, both enhancers must be present to modulate the IL-6-induced response which is diminished as a consequence of the 3′ enhancer mutation. These results suggest that the 3′ enhancer modulates the IL-6 response through an interaction with the 5′ enhancer. The mutation occurs at a DNA binding site for the ubiquitous transcription factor octamer-1 (Oct-1) and results in a loss of cooperativity between Oct-1 and the tissue-specific transcription factor, NF-IL6 (C/EBPβ), a member of the C/EBP family of transciption factors. NF-IL6 is a key transcription factor for a major pathway through which IL-6 mediates its effects. These observations provide a novel mechanism for a diminished IL-6-induced response.

Differential regulation of the mitochondrial ADP/ATP translocase gene in wood frogs under freezing stress

The gene Aat coding for ADP/ATP translocase (AAT) was cloned from liver of the freeze-tolerant wood frog, Rana sylvatica, via differential screening of a cDNA library from liver of frozen frogs and using probes from control versus frozen frogs. Sequence analysis showed that clone pBfFR07 bearing the AAT cDNA contained a 1318-bp insert with one full-length open reading frame. The deduced amino acid sequence included 317 residues, with 81–86% identities to mammalian AAT. A 1750-nt transcript from the Aat gene was detected using pBfFR07 probe and a putative frog AAT of over 30 kDa was visualized by immunoblotting using a polyclonal antibody raised against chicken AAT. Analysis of liver samples from a time course of freezing showed a maximal 4.5-fold increase in mRNA after 8 h with AAT protein peaking in 24-h frozen frogs. Freezing also induced Aat expression in bladder and lung. In liver, mRNA expression also responded positively to anoxia stress but not to experimental dehydration of the animals. These results suggest that AAT induction during freezing may be stimulated by the ischemia that develops when plasma freezes; changes in AAT may contribute to stabilizing energetics in mitochondrial versus cytosolic pools over freeze/thaw cycles.

The Aspergillus nidulans penicillin-biosynthesis gene aat (penDE) is controlled by a CCAAT-containing DNA element.

Analysis of the promoter of the penicillin biosynthesis aat (penDE) gene of Aspergillus nidulans using band-shift assays led to the identification of a CCAAT-containing DNA element which was specifically bound by a protein (complex). The identified DNA element was localised about 250 bp upstream of the transcriptional-start sites of aat. Substitution of the CCAAT core sequence by GATCC led to a fourfold reduction of expression of an aat-lacZ gene fusion. The identified binding site thus was functional in vivo and positively influenced at expression. Partial purification of the CCAAT binding protein and cross-competition experiments provided evidence that the binding protein is identical to the identified putative penicillin-regulatory protein PENR1, binding to the CCAAT element in the bidirectional intergenic promoter region between acvA (pcbAb) and ipnA (pcbC). Hence, PENR1 seems to be involved in the regulation of all three penicillin-biosynthesis genes. Cross-competition experiments demonstrated that the promoter region of the corresponding aat (penDE) gene of Penicillium chrysogenum was capable to dilute the shift of the A. nidulans probe with PENR1, suggesting the presence of a similar regulatory mechanism in this fungus. Taken together with previous data, CCAAT-containing DNA elements thus seem to represent major cis-acting sites in the promoters of beta-lactam-biosynthesis genes.

Long-term expression of the human α1-antitrypsin gene in mice employing anionic and cationic liposome vectors

The efficiency of both anionic and cationic liposomes as vectors for in vivo human α1-antitrypsin (AAT) gene transfer was studied in mice with and without an associated partial hepatectomy. The pTG7101 plasmid, containing the full-length human AAT gene, was encapsulated in small liposomes bearing 10% of negatively (phosphatidylserine, PS) or positively (DOTAP) charged lipids. The results indicate that the DNA/ lipid ratio was increased in cationic liposomes by inclusion of monosialoganglioside-G M1. The expression of human protein after in vivo gene transfer was quantified in mouse plasma by an ELISA procedure, and revealed that both anionic and cationic liposomes mediated the presence of human protein in mouse plasma for 2–3 weeks. This effect was prolonged (> 5 months) when a partial hepatectomy was performed after treatment. In addition, it was observed that the efficacy of liposome-mediated gene transfer was more limited when the plasmid was externally associated to cationic liposomes.

The Leucyl/Phenylalanyl-tRNA-Protein Transferase: OVEREXPRESSION AND CHARACTERIZATION OF SUBSTRATE RECOGNITION, DOMAIN STRUCTURE, AND SECONDARY STRUCTURE

Previous work has shown that, in the bacterium Escherichia coli, the aat gene is essential for the degradation of proteins bearing amino-terminal Arg and Lys residues via the N-end rule pathway of protein degradation. We now show that the aat gene encodes directly the leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase). This enzyme catalyzes the transfer of Leu, Phe, and, less efficiently, Met and Trp, from aminoacyl-tRNAs, to the amino terminus of acceptor proteins. We have used the cloned aat gene to overexpress and purify an affinity tagged L/F-transferase. The recombinant L/F-transferase is as active as the previously purified wild type enzyme and contains no detectable RNA component. We have used the recombinant enzyme to demonstrate that both the solubility and substrate specificity, for aminoacyl-tRNA substrates, of the L/F-transferase are dependent on ionic strength conditions and that the modified nucleotides found in natural tRNAs are not essential for recognition by the enzyme. Limited digestion of the L/F-transferase with trypsin removes the proline rich NH2 terminus of the enzyme identifying a globular core, and circular dichroism demonstrates that the L/F-transferase is predominantly alpha-helical. Finally, a region of sequence conservation between the L/F-transferase and the NH2-terminal protein acetylases has been identified.

Analysis of the regulation of the Aspergillus nidulans penicillin biosynthesis gene aat (penDE), which encodes acyl coenzyme A:6-aminopenicillanic acid acyltransferase.

The regulation of the Aspergillus nidulans penicillin biosynthesis gene aat (penDE), which encodes acyl coenzyme A:6-aminopenicillanic acid acyltransferase (AAT), was analysed. Major transcriptional start sites map within 100 nucleotides upstream from the aat initiation codon. To study the regulation of aat expression, various aat-lacZ gene fusions were constructed, in which the aat promoter region was fused in frame with the Escherichia coli lacZ reporter gene. A. nidulans strains carrying recombinant plasmids integrated as single copies at the chromosomal argB locus were identified. In both fermentation and minimal media, aat-lacZ expression was maximal during the first 24 h of a fermentation run. Compared with minimal medium, aat-lacZ expression was increased two-fold in fermentation medium. Although AAT specific activity was reduced in mycelia grown on glucose instead of lactose, expression of aat-lacZ gene fusions was not repressed on glucose, suggesting that the glucose effect is mediated posttranscriptionally. The effect of glucose on AAT activity was reversed by further incubation of glucose-grown mycelia on lactose. Neither the inclusion of the first intron of the aat gene in the aat-lacZ fusion integrated at the chromosomal argB locus, nor the disruption of the acvA gene had any regulatory effect on aat-lacZ expression. In the heterologous, nonpenicillin producer A. niger, basal expression of aat-lacZ gene fusions was observed at about the same level as in A. nidulans.