The gene encoding rat phosphoglycerate mutase subunit M: cloning and promoter analysis in skeletal muscle cells.

The expression of the gene encoding the muscle-specific (M)-subunit of phosphoglycerate mutase (PGAM-M) is restricted to adult skeletal and cardiac muscle. In order to study its expression in muscle, the rat PGAM-M gene has been isolated and sequenced. Rat PGAM-M spans about 2.2 kb and is composed of three exons: 442, 181 and 186-bp long, and two introns of 97 bp and 1.3 bp. The analysis of the 5'-flanking region reveals a promoter which contains multiple DNA regulatory elements and constitutes an ideal model to study muscle gene transcriptional regulation. Thus, the elements responsible for rat PGAM-M muscle-specific expression have been identified by transient transfection in chicken embryo primary cultures, using chimeric constructs of the rat promoter linked to a cat reporter gene. Here, we report that in spite of the abundance of E-box motifs in the rat PGAM-M promoter known for their involvement in muscle gene expression, two DNA elements regulate the muscle-specific transcription of rat PGAM-M: an A/T motif, the putative MEF-2-binding site (myocyte-specific enhancer-binding factor 2), and a proximal 27-bp element which is conserved between the rat and human genes. These two elements define a small promoter (170 bp) sufficient to support potent and skeletal-muscle-specific expression. The conserved 27-bp region contains a transcriptional regulatory element able to confer muscle-specific expression when located upstream from a heterologous TATA box.

Catalytic properties of recombinant 3-hydroxy-3-methylglutaryl coenzyme A synthase-1 from Blattella germanica.

Blattella germanica is the first organism in which two cytosolic HMG-CoA synthase genes have been described: HMGS-1 (Martínez-González et al., 1993b) and HMGS-2 (Buesa et al., 1994). The HMGS-1 gene showed special features, which led us to characterize the kinetic properties of the enzyme it encodes. Here we report the expression of recombinant HMGS-1, the protocol of enzyme purification, and the measurement of kinetic parameters. The K(m) for acetyl-CoA is 15.2 microM and the Ki for the other substrate, acetoacetyl-CoA, is 1.26 microM, both similar to that of yeast, ox, and chicken liver enzymes; the Vmax of HMGS-1 measured in this paper is 66 mU, which is the lowest Vmax of the HMG-CoA synthases reported to date.

3-Hydroxy-3-methylglutaryl coenzyme A synthase-1 of Blattella germanica has structural and functional features of an active retrogene.

Blattella germanica has two cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase genes, HMG-CoA synthase-1 and -2. HMG-CoA synthase-1 gene shows several features of processed genes (retroposons): it contains no introns but has a short direct-repeat sequence (ATTATTATT) at both ends. An atypical feature is the presence at both ends of the gene of short inverse repeats flanked by direct repeats. There is neither a TATA box nor a CAAT box in the 5' region. Comparative analysis with other species suggests that the HMG-CoA synthase-1 gene derives from HMG-CoA synthase-2. Cultured embryonic B. germanica UM-BGE-1 cells express HMG-CoA synthase-1 but not HMG-CoA synthase-2, suggesting that the intron-less gene is functional. In addition, it can complement MEV-1 cell line, which is auxotrophic for mevalonate. We show that compactin and mevalonate do not significantly affect the mRNA levels of HMG-CoA synthase-1 in UM-BGE-1 cells. Compactin induces a 6.7-fold increase in HMG-CoA reductase activity, which is restored to normal levels by mevalonate. HMG-CoA synthase activity is not modified by either of these effectors, suggesting that the mevalonate pathway in this insect cell line is regulated by post-transcriptional mechanisms affecting HMG-CoA reductase but not HMG-CoA synthase.

Coordinated expression and activity of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase in the fat body of Blattella germanica (L.) during vitellogenesis.

Levels of mRNA for the two 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthases, (HMG-S1 and HMG-S2), and for HMG-CoA reductase (HMG-R) of Blattella germanica were analyzed in the fat body during the first gonadotrophic cycle. HMG-S2 and HMG-R showed the highest mRNA levels on day 0 and decreased thereafter, whereas HMG-S1, showed faint expression. Western blot using specific antibodies for HMG-S1 and HMG-S2 showed no detectable levels for HMG-S1 but a clear pattern for HMG-S2. Both results point to a very limited role for HMG-CoA synthase-1 in B. germanica fat body that the functional enzyme in this organ is HMG-CoA synthase-2. HMG-CoA reductase and synthase proteins shared a cyclic pattern (maximum levels at day 4 and minimum levels on days 0 and 8), which was coincident with the pattern of activity. The delay between gene transcription and protein synthesis suggests a finely regulated translation mechanism. Moreover, the pattern of mevalonate synthesis parallels that of vitellogenin production, suggesting a coordinate mechanism between the mevalonate pathway and the production of vitellogenin.

Primary structure of scombrine alpha: two different species with an identical protamine.

We have studied the protamine scombrine alpha from the mackerel Scomber scombrus. Scombrine alpha is found phosphorylated in spermatid nuclei, but not in nuclei of ripe sperm. It is a typical fish protamine, made up of two distinct molecular species, each of 34 amino acid residues. The primary structure of the main component of scombrine alpha is 100% identical to scombrine gamma, the nonmicroheterogeneous protamine from Scomber australasicus (11). The second component of scombrine alpha is a very minor molecular species that has an isoleucine instead of a valine in position 11. Nuclear sperm-specific basic proteins display an enormous interspecific variability and it is very surprising that two different species show identical protamines. In this work we suggest that evolutionary changes in primary structure of protamines are restricted by several constitutive factors, especially when protamines either lack or have a low degree of microheterogeneity.

Lysosome-associated membrane protein 1 (LAMP-1) in Alzheimer's disease.

Lysosome-associated membrane protein 1 (LAMP-1) is a glycoprotein highly expressed in lysosomal membranes. The present study was initiated to test LAMP-1 mRNA and protein levels in post mortem frontal cortex (area 8) of Alzheimer's disease (AD) stages I-IIA/B and stages V-VIC of Braak and Braak, compared with age-matched controls. TaqMan PCR assays and Western blots demonstrated upregulation of LAMP-1 mRNA and protein in the cerebral cortex in ADVC. In addition, immunohistochemical studies have shown increased LAMP-1 immunoreactivity in neurones, and in glial cells surrounding senile plaques, in AD cases. Interestingly, LAMP-1 immunoreactivity has little correlation with phosphorylated tau deposition and neurofibrillary tangles (NFTs), as neurones with NFTs were rarely LAMP-1 immunoreactive. In contrast, LAMP-1 expression was enhanced in neurones with granulovacuolar degeneration. Finally, LAMP-1 occurred in microglia and multinucleated giant cells in one AD case in whom amyloid burden was cleared following betaA-peptide immunization. These findings support the participation of lysosomes in betaA-amyloid and, probably, in hyperphosphorylated tau turnover in AD.

3-Hydroxy-3-methylglutaryl-coenzyme-A synthase from Blattella germanica. Cloning, expression, developmental pattern and tissue expression.

Insects do not synthesize cholesterol; the 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) produced by HMG-CoA synthase is transformed to mevalonate by HMG-CoA reductase for the production of non-sterol isoprenoids, which are essential for growth and differentiation. To understand the regulation and developmental role of HMG-CoA synthase, we have cloned a 1658 bp cDNA that encompasses the entire transcription unit of the HMG-CoA synthase gene from the cockroach Blattella germanica. This cDNA clone was isolated using as a probe a partial cDNA of B. germanica HMG-CoA synthase, amplified using the polymerase chain reaction. Analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 453 amino acids (M(r) 50338) that is similar to vertebrate HMG-CoA synthase (74-76% conserved residues). The B. germanica cDNA has been expressed as a fusion protein in Escherichia coli and exhibits HMG-CoA synthase activity. The HMG-CoA synthase transcript was differentially expressed throughout B. germanica development. Analysis of RNA samples from different adult female tissues shows high HMG-CoA synthase mRNA levels in the ovary and lower levels in brain and muscle.

The primary structure of a chondrichthyan protamine: a new apparent contradiction in protamine evolution.

We have determined the primary structure of protamine R3 from ratfish (Hydrolagus colliei), a species belonging to the order Chimaeriformes (an old phylogenetic line among the chondrichthyes). Protamine R3 contains 48 residues organized as follows: ARRRH SMKKK RKSVR RRKTR KNQRK RKNSL GRSFK (Q/A)HGFL KQPPR FRP. Comparison of this sequence with both protamine Z3 from Scyliorhinus canicula (a chondrichthyan) and typical protamines from bony fish generates an apparent contradiction: Two relatively close species (H. colliei and S. canicula, both chondichthyes) display different protamines, whereas species more distant in evolution (S. canicula and bony fish) contain very similar protamine molecules. We note that this is not an isolated case in the evolution of sperm nuclear basic proteins (SNBPs) and discuss the possible significance of this fact.

Differences in chromatin condensation during spermiogenesis in two species of fish with distinct protamines.

The sperm cells of Mullus surmuletus (family Mullidae, order Perciformes) and Dicentrarchus labrax (family Percichthyidae, order Perciformes) belong respectively to "type I" and "type II" spermiogenesis categorized by Mattei ('70). The protein content in their sperm nuclei consists of two histone-like proteins (Mullus surmuletus) and one typical protamine (D. labrax). In order to correlate the molecular characteristics of these proteins with their function, we have analyzed the molecules in detail and studied at the ultrastructural level the condensation of chromatin during the spermiogenesis in both species. D. labrax has a true protamine of 34 amino acid residues and its sequence (PR4QASRPVR5TR2STAER5V2R4) contains four arginine clusters. The sperm proteins of M. surmuletus contain 110 and 115 amino acid residues and , by their composition (23-24% Lys, 21-22% Arg, 11-12% Ala), they are similar to protamine-like molecules from sperm of molluscs. During the spermiogenesis of D. Labrax, chromatin condensation progresses from small fibro-granular structures (25 +/- 5 nm in diameter), to larger granules (150 +/- 50 nm diameter). M. surmuletus accumulates 25 +/- 5 nm diameter structures in the basal pole of the nucleus; these structures grow till they reach a diameter of 50 +/- 10 nm and finally go through a process of fusion that changes the condensation of chromatin in sperm nuclei, acquiring a homogeneous aspect. These observations show that during spermiogenesis in the studied types, the last stages of chromatin condensation are dependent on the type of nuclear proteins.

Molecular cloning and tissue expression of human mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase.

Mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase is a constituent of the HMG-CoA pathway responsible for ketone body synthesis and one of its main regulatory points. We report the isolation and characterization of a 2058-bp cDNA from human liver. This cDNA encodes a polypeptide of 508 residues and 56,635 Da. The homology with previously reported rat mitochondrial and human cytosolic HMG-CoA synthases is 88 and 66%, respectively. mRNA levels were high in liver and colon, low in testis, heart, skeletal muscle, and kidney, and faint in pancreas.

Molecular cloning, developmental pattern and tissue expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase of the cockroach Blattella germanica.

In insects, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) synthesizes mevalonate for the production of nonsterol isoprenoids, which are essential for growth and differentiation. To understand the regulation and developmental role of HMG-CoA reductase, we have cloned a full-length HMG-CoA reductase cDNA from the cockroach Blattella germanica. This cDNA clone was isolated using as a probe a partial cDNA of B. germanica HMG-CoA reductase, amplified using the polymerase chain reaction. The composite 3433-bp cDNA sequence contains an open reading frame encoding a polypeptide of 856 amino acids (Mr, 93165). The C-terminal region is more similar to hamster HMG-CoA reductase than is the Drosophila melanogaster enzyme (79% and 69% conserved residues, respectively), and the potential transmembrane domains at the N-terminal region are structurally conservative with both enzymes. The C-terminal region of the B. germanica protein has been expressed as a fusion protein in Escherichia coli and exhibits HMG-CoA reductase activity. Analysis of B. germanica HMG-CoA reductase mRNA levels, reveals a 3.6-kb transcript, that is overexpressed in 4-day-old embryos. Northern-blot analysis of RNA samples from different adult female tissues shows high HMG-CoA reductase mRNA levels in the ovary and lower levels in brain and muscle.

Purification and characterization of a new alpha-amylase of intermediate thermal stability from the yeast Lipomyces kononenkoae.

A new alpha-amylase from the extracellular culture of the yeast Lipomyces kononenkoae CBS 5608 has been purified to homogeneity by ammonium sulphate treatment, affinity binding on cross-linked starch, and DEAE-Biogel A chromatography. The enzyme was monomeric, with an apparent M(r) of 76 kilodaltons, pI < 3.5, and optimum pH 4.5-5.0, and exhibited intermediate thermal stability. The temperature for optimal enzyme activity was 70 degrees C. It is a glycoprotein with both N- and O-linked sugars. Kinetic analyses indicate that the enzyme has an endoamylolytic mechanism. The kM for soluble starch was 0.80 g.L-1 and the kcat was 622.s-1.

Blattella germanica has two HMG-CoA synthase genes. Both are regulated in the ovary during the gonadotrophic cycle.

The isoprenoid pathway leads to various essential non-sterol products in insects. These end products have a crucial role in growth, differentiation, sexual maturation, and reproduction. 3-Hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) synthase (EC 4.1.3.5.) has generally been considered one of the committed steps of the pathway. We had previously reported the cloning of a cytosolic HMG-CoA synthase cDNA in Blattella germanica; we have now isolated and characterized a new cDNA clone for HMG-CoA synthase in this insect. Analysis of this 1716-base pair cDNA reveals a deduced protein of 455 residues with a molecular mass of 51,424 Da. The two HMG-CoA synthases have 69% identical amino acid residues, and both lack an N-terminal leader peptide to target the protein into mitochondria. This HMG-CoA synthase cDNA can revert the Chinese hamster ovary-K1-derived cell line, Mev-1, which is a defective mutant for HMG-CoA synthase. Both HMG-CoA synthase genes are expressed differently throughout development. Analysis of adult tissues shows higher expression in ovary and fat body. The expression of HMG-CoA synthase (EC 4.1.3.5.) and reductase (EC 1.1.1.34) genes during the gonadotrophic cycle in B. germanica shows that the three genes of the isoprenoid pathway are developmentally regulated in the ovary.

Aberrantly spliced mRNAs of the 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL) gene with a donor splice-site point mutation produce hereditary HL deficiency.

A novel point mutation in the 3-hydroxy-3methyl-glutaryl coenzyme A lyase gene was found in a Turkish patient with homozygous 3-hydroxy-3-methylglutaric acidemia. Amplification by RT-PCR of the mRNA using a six different pairs of oligonucleotides produced no differences in four of the fragments amplified with respect to the control, but generated two fragments of different size. One was representative of a deletion of 126 bp and the other of an insertion of 78 bp. These abnormal mRNAs resulted from a G-->C transversion at the nucleotide +1 of an intron, which changed the invariant GT dinucleotide of the 5' donor splice site. This was associated with the occurrence of an alternative splicing, which led to the skipping of the whole exon of 126 bp, and also with the activation of one cryptic donor splice site in the same intron. These aberrant spliced mRNAs are predicted to encode two abnormal HMG-CoA lyase proteins: the first results in a protein with an internal deletion of 42 amino acids, whose enzyme activity is largely abolished, as the catalytic site was completely removed; the second contains 17 missense amino acids that precede a stop codon. Northern blot analysis showed that the overall content of these aberrantly spliced mRNAs in proband fibroblasts was the same as that found in control fibroblasts. However, hardly any transcript was observed corresponding to the inserted mutated mRNA when it was examined by a specific probe. To quantify the relative proportion of the two mRNAs, a quantitative RT-PCR (the DNA-mimic PCR reaction) was carried out. Results show that the proportion of the inserted mRNAs with respect to the deleted mRNA is only 1.2%. The father, mother, and two brothers of the proband were heterozygous in the G-->C mutation in the +1 nucleotide of the intron considered, while the two alleles of another brother were free of the mutation.

A nonsense mutation in the 3-hydroxy-3-methylglutaryl-CoA lyase gene produces exon skipping in two patients of different origin with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

A novel nonsense mutation associated with the skipping of constitutive exon 2 of the 3-hydroxy-3-methylglutaryl-CoA lyase gene was found in two patients, from Portugal and Morocco, with 3-hydroxy-3-methylglutaric acidemia. By reverse transcriptase PCR and single-strand conformational polymorphism a G-T transversion was located, at nucleotide 109, of the 3-hydroxy-3-methylglutaryl-CoA lyase cDNA, within exon 2. Two mRNAs were produced as a result of this nonsense mutation: one of the expected size that contains the premature stop codon UAA, and the other with a deletion of 84 bp corresponding to the whole of exon 2. This deletion produced the loss of the last seven amino acids of the leader peptide and the first 21 amino acids of the mature protein. The nonsense mutation was found in a purine-rich GGAAG sequence, which is equal to, or similar to, others reported to be exonic splicing enhancers (ESE). We suggest that the nonsense mutation may affect a possible ESE on exon 2, which would hinder the splice site selection and facilitate an aberrant splice with the skipping of this exon. Determination by quantitative PCR shows that the ratio of mRNA with the nonsense mutation to the mRNA with the deletion is approx. 3:1.