Biomedical subjects

M J Hynes

Publications and source records attributed to M J Hynes.

At least 55 records · Page 3Linked to original sources

PubMed1993

Fungal catabolic gene regulation: molecular genetic analysis of the amdS gene of Aspergillus nidulans.

Aspergillus nidulans is an excellent experimental organism for the study of gene regulation. Genetic and molecular analyses of trans-acting and cis-acting mutations have revealed a complex pattern of regulation involving multiple independent controls. Expression of the amdS gene is regulated by the facB and amdA genes which encode positively acting regulatory proteins mediating a major and a minor form of acetate induction respectively. The product of the amdR gene mediates omega amino acid induction of amdS. The binding sites for each of these proteins have been localised through amdS cis-acting mutations which specifically affect the interaction with the regulatory protein. The global controls of nitrogen metabolite repression and carbon catabolite repression regulate the expression of many catabolic genes, including amdS. Nitrogen control is exerted through the positively acting areA gene product and carbon control is dependent on the creA gene product. Each of the characterized regulatory genes encodes a DNA-binding protein which recognises particular sequences in the amdS promoter to activate or repress gene expression. In addition, there is evidence for other genetically uncharacterized proteins, including a CCAAT-binding complex, which interact with the 5' region of the amdS gene.

Amidohydrolases↗

PubMed1992

Structural and functional analysis of the amdR regulatory gene of Aspergillus oryzae.

We have isolated the Aspergillus oryzae homologue of the amdR regulatory gene of Aspergillus nidulans by cross hybridization. Sequence analysis and functional studies have shown that the amdR genes are highly conserved and functionally interchangeable between the two species. The homology between the two genes extends throughout most of the coding sequences, including sequences encoding the DNA-binding domain and putative activation domains. Two regions of nonconserved sequence were also identified. Studies using various amdS::lacZ fusion constructs indicate that the A. oryzae gene product binds similar sequences and responds to inducer in a similar manner to the A. nidulans protein. Inactivation of the A. oryzae gene results in the inability to grow on gamma-amino-butyric acid (GABA) as a carbon and/or nitrogen source indicating that GABA utilization is amdR-dependent in A. oryzae as it is in A. nidulans.

Amino Acid Sequence↗

PubMed1992

Characterization of the amdA-regulated aciA gene of Aspergillus nidulans.

The structure, function and regulation of the acetate inducible aciA gene of Aspergillus nidulans was analysed. The aciA locus was mapped to chromosome 1 at a position where no acetate inducible gene has been previously located. The nucleotide sequence of aciA was determined, the structures of two transcripts were determined and the sequences of the polypeptide products of the gene were deduced. Construction of an aciA loss-of-function mutant was achieved via insertional inactivation, but it did not reveal a phenotype for an aciA- strain. The larger polypeptide, AciA, was found to have a putative dinucleotide cofactor binding site. Acetate inducibility of aciA was found to be dependent on the amdA regulatory gene. Use of a 5' deletion series of an aciA--lacZ fusion and an in vivo regulatory protein binding (titration) assay allowed the region required for amdA activity to be localized to a 124 bp segment 5' to aciA. A 13 bp region of sequence similarity was observed between aciA and the coregulated amdS gene in the regions required for amdA-mediated regulation of these genes. This sequence may have a role in amdA regulation of the two genes.

Amino Acid Sequence↗

PubMed1992

Analysis of the site of action of the amdR product for regulation of the amdS gene of Aspergillus nidulans.

The amdR gene of Aspergillus nidulans regulates a number of structural genes in response to omega amino acid inducers. The site of action of the amdR product on expression of the amdS gene was investigated by studying the effects of changes in the 5' region of amdS, generated in vitro, on the induction, and on responses of an amdS-lacZ fusion gene to an amdRc allele. A sequence was identified that is sufficient for amdR regulation and that shows identity with sequences involved in amdR regulation of the gatA and lam genes. This sequence includes a CCAAT sequence and it was shown that this sequence is an important element in setting the basal level of amdS expression.

Amidohydrolases↗

PubMed1992

Identification of functional regions of the positively acting regulatory gene amdR from Aspergillus nidulans.

The amdR (intA) regulatory gene of Aspergillus nidulans encodes a 765-amino-acid polypeptide which determines the omega-amino acid induction of at least five structural genes. The AmdR polypeptide contains a potential Zn(II)2Cys6 DNA-binding motif which has been shown to be present in the N-terminal region of a large number of fungal activator proteins. In vitro mutagenesis of the fourth cysteine of this motif abolishes AmdR function as shown by loss of complementation of an amdR- mutation and by the AmdR- phenotype of a mutant gene replacement strain. Studies using constructs in which the proposed AmdR DNA-binding motif is replaced with that from another activator, FacB, shows that induction is independent of DNA-binding specificity and that sequences in the C-terminal region of AmdR are activation domains. Sequencing of several amdR mutant alleles which affect activation and/or induction, together with studies of deletion constructs indicate that changes in the conformation of the protein determines its activity and that this is modulated by inducers.

Aspergillus nidulans↗

PubMed1992

Molecular characterization of the lam locus and sequences involved in regulation by the AmdR protein of Aspergillus nidulans.

The lam locus of Aspergillus nidulans consists of two divergently transcribed genes, lamA and lamB, involved in the utilization of lactams such as 2-pyrrolidinone. Both genes are under the control of the positive regulatory gene amdR and are subject to carbon and nitrogen metabolite repression. The lamB gene and the region between the two genes have been sequenced, and the start points of transcription have been determined. Within the lam locus are two sequences with homology to elements, required for AmdR regulation, found in the 5' regions of the coregulated genes amdS and gatA. In vitro and in vivo assays were used to investigate the lam and gatA regulatory elements. One of the three gatA elements and one of the two lam elements were shown to bind AmdR protein in vivo and activate transcription. With a gel shift mobility assay, in vitro binding of AmdR protein to the functional gatA element was detected. Both the functional gatA and lam boxes contain within them a CAAT sequence. In vitro binding analysis indicates that a CCAAT-specific factor(s) binds at these sequences, adjacent to or overlapping the AmdR protein-binding site.

Amino Acid Sequence↗

PubMed1991

The amdR product and a CCAAT-binding factor bind to adjacent, possibly overlapping DNA sequences in the promoter region of the Aspergillus nidulans amdS gene.

The amdS gene of Aspergillus nidulans is regulated by a number of positively acting regulatory genes which act additively and independently. Using gel mobility shift assays with crude nuclear extracts we show here that the product of one of these regulatory genes, the amdR gene, binds to DNA fragments containing part of the promoter region of the amdS gene. This confirms the earlier prediction from DNA sequence data that amdR encodes a DNA-binding protein containing a cysteine-rich 'zinc finger' motif. In addition we detected the binding of another previously unidentified protein to an adjacent, possibly overlapping region of the amdS 5' sequence at the site of a consensus 'CCAAT-box' sequence. Replacement of the CCAAT sequence with CCTTT abolished the binding of this protein which we have designated as an A. nidulans 'CCAAT-box' binding factor (AnCF). The 'CCAAT-box' sequence appears to be involved in determining the basal level of transcription of amdS (T.G. Littlejohn and M.J.H., unpublished data). This suggests that AnCF is a transcription factor, and that the 'CCAAT-box' sequences found in the promoters of some filamentous fungal genes function as binding sites for these factors, as in other eucaryotes.

Amidohydrolases↗

PubMed1991

Molecular organisation of the malate synthase genes of Aspergillus nidulans and Neurospora crassa.

The sequencing and comparison of the genes encoding the glyoxylate bypass enzyme malate synthase of Aspergillus nidulans (acuE) and Neurospora crassa (acu-9) are presented. The predicted amino acid sequences of the A. nidulans and N. crassa enzymes are 538 and 542 residues respectively and the proteins are 87% homologous. In fungi, the malate synthase proteins are located in glyoxysomes and the deduced acuE and acu-9 proteins both contain a C-terminal S-K-L sequence, which has been implicated in transport into peroxisomes. The acuE coding region is interrupted by four introns and the acu-9 coding region is interrupted by one intron which occurs at the same position as the C-terminal acuE intron. The 5' non-coding regions of the two genes were examined for short homologous sequences that may represent the binding sites for regulatory proteins. Pyrimidine-rich sequences with weak homology to the amdI9 sequence, which has been implicated in facB-mediated acetate regulation of the amdS gene, were found but their functional significance remains to be determined.

Amino Acid Sequence↗

PubMed1990

A mutation affecting amdS expression in Aspergillus nidulans contains a triplication of a cis-acting regulatory sequence.

In Aspergillus nidulans expression of the acetamidase structural gene, amdS, is under the control of at least four regulatory genes including the trans-acting amdA regulatory gene. A cis-acting mutation (amdI66) consisting of an 18 bp duplication in the 5' region of the amdS gene results in very high levels of acetamidase activity but only in strains carrying semi-dominant mutations in the amdA gene. In selecting for increased amdS expression in an amdI66 and A+ strain, an A. nidulans strain with a mutation in the 5' region of the amdS gene was isolated. The nucleotide sequence was determined of the region containing the mutation, designated amdI666. The mutant strain carries three tandem copies of the 18 bp sequence that is duplicated in the amdI66 mutation. Thus, from a strain carrying a duplication of an apparent regulatory protein binding site with little effect on gene expression, a strain has been derived that carries a triplication of the site with consequent major effects on regulation. The multiple copies of regulatory sites present in many genes may have been generated by a similar mechanism.

Amidohydrolases↗

PubMed1990

Comparison and cross-species expression of the acetyl-CoA synthetase genes of the Ascomycete fungi, Aspergillus nidulans and Neurospora crassa.

The genes encoding the acetate-inducible enzyme acetyl-coenzyme A synthetase from Neurospora crassa and Aspergillus nidulans (acu-5 and facA, respectively) have been cloned and their sequences compared. The predicted amino acid sequence of the Aspergillus enzyme has 670 amino acid residues and that of the Neurospora enzyme either 626 or 606 residues, depending upon which of the two possible initiation codons is used. The amino acid sequences following the second alternative AUG show 86% homology between the two species; the extended N-terminal sequences show no homology. The Neurospora protein is characterized by the appearance of the S(T)PXX sequence motif where the amino acid homologies break down. The codon usage is biased in both genes, with a marked deficiency, especially in Neurospora, of codons with A in the third position. The facA transcribed sequence contains six introns: one in the long leader sequence, one in the 5' coding sequence not homologous with acu-5, and four within the sequence that is largely similar to that of acu-5. Only one intron, corresponding in size and position to the furthest downstream of the facA introns, is found in acu-5. The evolution of introns during the divergence of these two Ascomycete fungi is discussed. Each of the two genes has been transferred by transformation into the other species. Each species is evidently able to splice out the other's introns. Most transformants have normal acetate-induction of acetyl-CoA synthetase, implying that the two genes respond to transcriptional control signals common to both species, in spite of the striking divergence of their 5' ends.

Acetate-CoA Ligase↗

PubMed1990

Expression and secretion in Aspergillus nidulans and Aspergillus niger of a cell surface glycoprotein from the cattle tick, Boophilus microplus, by using the fungal amdS promoter system.

A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus, which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed and secreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promoter system. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide except for the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformed cells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to 87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditions used. No intracellular Bm86 was detected. A general relationship was observed between transformants containing a high number of copies of the expression plasmid and high expression levels.

Animals↗

PubMed1990

Sequence and functional analysis of the positively acting regulatory gene amdR from Aspergillus nidulans.

The positively acting regulatory gene amdR of Aspergillus nidulans coordinately regulates the expression of five structural genes involved in the catabolism of certain amides (amdS), omega amino acids (gatA and gabA), and lactams (lamA and lamB) in the presence of omega amino acid inducers. Analysis of the amdR gene showed that it contains three small introns, heterogeneous 5' and 3' transcription sites, and multiple AUG codons prior to the major AUG initiator. The predicted amdR protein sequence has a cysteine-rich "zinc finger" DNA-binding motif at the amino-terminal end, four putative acidic transcription activation motifs in the carboxyl-terminal half, and two sequences homologous to the simian virus 40 large T antigen nuclear localization motif. These nuclear localization sequences overlap the cysteine-rich DNA-binding motif. A series of 5', 3', and internal deletions were examined in vivo for transcription activator function and showed that the amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator regions may function independently, but both are required for wild-type levels of transcription activation. A number of the amdR deletion products were found to compete with the wild-type amdR product in vivo. Development of a rapid method for the localization of amdR mutations is presented, and using this technique, we localized and sequenced the mutation in the semiconstitutive amdR6c allele. The amdR6c missense mutation occurs in the middle of the gene, and it is suggested that it results in an altered protein which activates gene expression efficiently in the absence of an inducer.

Amino Acid Sequence↗

PubMed1989

Gene function identified by interspecific transformation.

Aspergillus nidulans is able to utilize 2-pyrrolidinone as a nitrogen source while two related Aspergillus species, A. niger and A. terreus, cannot. Mutations in the lamA gene of A. nidulans prevent growth on 2-pyrrolidinone. A plasmid (pLAM7) has been isolated containing the A. nidulans lamA gene and a divergently transcribed adjacent gene of unknown function. Transformation of A. terreus with subclones of pLAM7 showed that both genes are essential for the utilization of a new nitrogen source, 2-pyrrolidinone, in that species. The previously unidentified gene has been designated lamB.

Aspergillus↗

PubMed1989

Isolation of the facA (acetyl-coenzyme A synthetase) and acuE (malate synthase) genes of Aspergillus nidulans.

Acetate inducible genes of Aspergillus nidulans were cloned via differential hybridization to cDNA probes. Using transformation of mutant strains the genes were identified as facA (acetyl-Coenzyme A synthetase) and acuE (malate synthase). The levels of RNA encoded by these genes were shown to be acetate inducible and subject to carbon catabolite repression. Induction is abolished in a facB mutant and carbon catabolite repression is relieved in a creA mutant.

Acetate-CoA Ligase↗

PubMed1989

Cloning and molecular characterisation of the amdR controlled gatA gene of Aspergillus nidulans.

The gamma-amino-n-butyrate transaminase gene (gatA) of Aspergillus nidulans is one of several genes under positive control by the regulatory gene amdR (also called intA). The gatA gene has been cloned from a cosmid library by complementation of a gatA mutation. The sequence of a 2.6 kb genomic fragment containing gatA has been determined. An open reading frame of 1497 bp within this sequence is interrupted by three putative introns and predicts a protein of 55 kDa. Northern analysis confirms control of gatA RNA levels by amdR and also indicates that gatA is not strongly regulated by areA-mediated nitrogen metabolite repression. A. nidulans transformants containing multiple copies of a plasmid carrying an 88 bp fragment from the 5' untranscribed region of gatA grew poorly on substrates whose utilisation is dependent on genes controlled by amdR. This indicated titration of limiting amounts of the amdR gene product by this 88 bp fragment. Comparison of this sequence with the 5' region of the coregulated gene, amdS, reveals probable sites of action for the amdR protein.

4-Aminobutyrate Transaminase↗

PubMed1989

Regulatory genes in Aspergillus nidulans.

A major area for the study of gene regulation in lower eukaryotes has been the coordinated control of catabolic enzyme synthesis. Studies of catabolic gene regulation aim to define how interactions between input signals and regulatory proteins are transmitted to the transcription machinery to bring about changes in gene expression. In the past, mutants altered in the utilization of a wide variety of substrates have been characterized in Aspergillus nidulans. Recently, the development of a transformation system for A. nidulans has meant that molecular techniques can now be combined with the traditional genetic approach.

Aspergillus nidulans↗

PubMed1989

Characterization of the amdR-controlled lamA and lamB genes of Aspergillus nidulans.

Four Aspergillus nidulans genes are known to be under the control of the trans-acting regulatory gene amdR. We describe the isolation and initial characterization of one of these amdR-regulated genes, lamA. The lam locus, however, was found to consist of two divergently transcribed genes, the lamA gene, and a new gene, also under amdR control, which we have designated lamB. Using recombinant DNA techniques we have constructed a strain of A. nidulans lacking a functional lamB gene. Experiments conducted with this strain demonstrate that lamB, like lamA, is involved in utilization of 2-pyrrolidinone in A. nidulans. Metabolism of a related compound, gamma-amino butyric acid (GABA) is not affected. We also provide evidence that the conversion of exogenous 2-pyrrolidinone to endogenous GABA requires a functional lamB gene. The expression of both lamA and lamB is subject to carbon and nitrogen metabolite repression in addition to amdR-mediated induction by omega-amino acids.

Aspergillus nidulans↗

PubMed1989

Patient age and success in a human IVF programme.

Previous studies suggest that at around 40 years of age, pregnancy rates achieved by IVF programmes fall and pregnancy loss rates increase. The actual age at which this occurs has not been clearly delineated. This study of 2,692 patients including 94 aged 41 or over shows that satisfactory pregnancy rates can be achieved up to and including age 40. As age 40 is approached the pregnancy loss rate increases to around 40%. In the 41 years and over group the pregnancy rate was poor at 6% (6/94) and the pregnancy loss rate very discouraging at 83% (5/6).

Adult↗