Analysis of dystrophin gene deletions indicates that the hinge III region of the protein correlates with disease severity.

We have investigated the frequency of deletions in the dystrophin gene in 108 unrelated Duchenne and Becker muscular dystrophy (DMD/BMD) patients from southern Italy (DMD, n. 47; BMD, n. 61) and identified 89 deletions. The de novo mutation rate (about 30%), and the preferentially maternal origin of deletional mutations, analysed in families in which the maternal grandparents were available or their haplotypes could be unequivocally reconstructed, are in agreement with data reported for other populations. The correlation between BMD phenotype and type of deletion suggests that, in the distal rod domain region, the deletion size may not be as crucial as the particular combination of missing exons. In fact, we provide immunohistochemical and clinical evidence that in-frame deletion of the hinge III region in the distal rod domain results in a milder phenotype as compared with shorter deletions that do not include the hinge III region. Our data obtained in BMD patients, by confirming inferences arising from minigene transfection experiments in mdx mice, represent an important contribution to gene therapy approaches.

Recent advances in the diagnosis of malignant hyperthermia susceptibility: how confident can we be of genetic testing?

Malignant hyperthermia (MH) is a condition that manifests in susceptible individuals only on exposure to certain anaesthetic agents. Although genetically heterogeneous, mutations in the RYR1 gene (19q13.1) are associated with the majority of reported MH cases. Guidelines for the genetic diagnosis for MH susceptibility have recently been introduced by the European MH Group (EMHG). These are designed to supplement the muscle biopsy testing procedure, the in vitro contracture test (IVCT), which has been the only means of patient screening for the last 30 years and which remains the method for definitive diagnosis in suspected probands. Discordance observed in some families between IVCT phenotype and susceptibility locus genotype could limit the confidence in genetic diagnosis. We have therefore assessed the prevalence of 15 RYR1 mutations currently used in the genetic diagnosis of MH in a sample of over 500 unrelated European MH susceptible individuals and have recorded the frequency of RYR1 genotype/IVCT phenotype discordance. RYR1 mutations were detected in up to approximately 30% of families investigated. Phenotype/genotype discordance in a single individual was observed in 10 out of 196 mutation-positive families. In five families a mutation-positive/IVCT-negative individual was observed, and in the other five families a mutation-negative/IVCT-positive individual was observed. These data represent the most comprehensive assessment of RYR1 mutation prevalence and genotype/phenotype correlation analysis and highlight the possible limitations of MH screening methods. The implications for genetic diagnosis are discussed.

Identification of a novel mutation in the ryanodine receptor gene (RYR1) in a malignant hyperthermia Italian family.

Malignant hyperthermia (MH) is an inherited autosomal dominant pharmacogenetic disorder and is one of the main causes of death subsequent to anaesthesia. Around 50% of affected families are linked to the ryanodine receptor (RYR1) gene. To date, 19 mutations have been identified in the coding region of this gene and appear to be associated with the MH-susceptible phenotype. Here we report the identification by two independent methods of a novel mutation associated with the MH-susceptible phenotype in the RYR1 gene: the 6488G-->C transversion, resulting in the replacement of the Arg2163 with a proline residue.

A case of discordance between genotype and phenotype in a malignant hyperthermia family.

Malignant hyperthermia (MH) is an inherited autosomal dominant pharmacogenetic disorder and is the major cause of anaesthesia-induced death. Malignant hyperthermia susceptibility is usually diagnosed by the in vitro contracture test (IVCT) performed on fresh muscle biopsies exposed to caffeine and halothane, respectively. Around 50% of affected families are linked to the ryanodine receptor (RYR1) gene. The human RYR1 gene maps to chromosome 19q13.1 and encodes a protein that associates as a homotetramer and acts as a calcium-release channel from the sarcoplasmic reticulum. To date, 17 mutations have been identified in the coding region of the RYR1 gene and appear to be associated to the MH-susceptible phenotype. Here we describe a rare case of discordance between genotype (characterised by the presence of the Arg614Cys mutation in the RYR1 gene) and MH-normal typed phenotype. Although the IVCT remains a very reliable procedure for the assessment of MH status, genetic data can provide in some cases an additional aid to clinical diagnosis.

The differential pattern of tissue-specific expression of ruminant pancreatic type ribonucleases may help to understand the evolutionary history of their genes.

Molecular evolutionary analyses of mammalian ribonucleases have shown that gene duplication events giving three paralogous genes occurred in ruminant ancestors. The enzymes of the bovine species encoded by these genes, isolated from pancreas, brain and seminal vesicles, present similar enzymological properties but distinct structural features. In other ruminant species, genomic sequences orthologous to the bovine genes of pancreas and brain ribonucleases encode active enzymes. In mammalian species other than ruminant artiodactyls, only one gene encoding ribonuclease of the pancreatic type is generally present. In this work, we describe a differential pattern of transcriptional expression of the pancreas and brain ribonuclease genes in the ox species and report transcription of the human ribonuclease gene in brain as well as in pancreas and in mammary gland. We also report the molecular cloning of the gene encoding the bovine seminal ribonuclease in which the structural organization already described for the two paralogous genes is conserved. The seminal RNAase is exclusively expressed in seminal vesicles of Bos taurus, whereas in other ruminant species, the orthologous sequence is a pseudogene. Previous studies from a number of research groups demonstrated that, unlike other mammalian ribonucleases, the seminal enzyme is a covalent dimer, and its unique quaternary structure correlates with special biological activities. The major determinant of dimer formation, i.e. the presence of two adjacent cysteine residues, is absent in the pseudogenes. We advance the hypothesis that the differentiation of distinct expression patterns could represent an important evolutionary determinant for the genes encoding pancreas and brain ribonucleases in ruminants, whereas the differentiation of a quaternary structure endowed with new biological functions could be the main determinant for the evolutionary success of the seminal gene in the bovine species.

Lithostathine messenger RNA expression in different types of chronic pancreatitis.

Lithostathine may play a physiological role in preventing the precipitation of excess calcium in the pancreatic juice. The hypothesis has been advanced that in chronic calcifying pancreatitis the abnormal biosynthesis of lithostathine might be the original defect to which genetic proneness to the disease may be ascribed. The aim of the present work was to study lithostathine messenger RNA expression in the pancreas of patients with different types of pancreatitis. Lithostathine and chymotrypsinogen mRNA were determined in surgical specimens obtained from the pancreases of the following subjects: (a) 13 patients with chronic alcoholic pancreatitis (84.6% calcified); (b) 4 patients with chronic hereditary pancreatitis (all calcified); (c) 6 patients with chronic obstructive pancreatitis (4 calcified); and (d) 27 subjects suffering from pancreatic cancer. Significantly lower concentrations of both mRNAs were found in the pancreases of chronic pancreatitis patients than in non-cancerous tissue from pancreatic cancer subjects. However, about 70% of the pancreatic cancer subjects showed lithostathine and chymotrypsinogen mRNA levels comparable to those of chronic pancreatitis patients. These results indicate that the decrease in the level of mRNA is not specific to lithostathine and it is unrelated to the presence of pancreatic stones.

Nuclear factor kappa B (NF-kappa B), nuclear factor interleukin-6 (NFIL-6 or C/EBP beta) and nuclear factor interleukin-6 beta (NFIL6-beta or C/EBP delta) are not sufficient to activate the endogenous interleukin-6 gene in the human breast carcinoma cell line MCF-7. Comparative analysis with MDA-MB-231 cells, an interleukin-6-expressing human breast carcinoma cell line.

A comparative study of the molecular mechanism of interleukin-6 (IL-6) gene induction on two breast-carcinoma-derived cell lines has been performed. MDA-MB-231 cells produce constitutive detectable levels of both secreted IL-6 and mRNA which, as expected, are dramatically enhanced following induction by either IL-1 beta or tumor necrosis factor-alpha (TNF-alpha). The levels of both secreted IL-6 and IL-6 mRNA are significantly higher in response to IL-1 beta in spite of the fact that stimulation by TNF-alpha alone enhances the half life of IL-6 mRNA. The protein synthesis inhibitor cycloheximide is also a fairly strong inducer of IL-6 in these cells. In contrast, MCF-7 cells fail to produce detectable IL-6 protein or mRNA, even after stimulation with proper inducers. Analysis of transcription factors NF-kappa B, NFIL6 and NFIL6 beta, which have been described to be sufficient to activate the IL-6 gene in other cell systems, shows a similar pattern of expression in both MCF-7 and MDA-MB-231 cells. Furthermore, transfection of a recombinant plasmid carrying the IL-6 promoter linked to a luciferase reporter gene shows that both cell lines are able to drive IL-1 beta or TNF-alpha activation of this construction in a very similar manner. Finally, when MCF-7 cells were treated with IL-1 beta or TNF-alpha in the presence of cycloheximide, transcription of IL-6 mRNA from the endogenous IL-6 gene was observed. These data suggest that a mechanism of IL-6 gene repression is active in MCF-7 cells.

Molecular evolution of genes encoding ribonucleases in ruminant species.

Phylogenetic analysis, based on the primary structures of mammalian pancreatic-type ribonucleases, indicated that gene duplication events, which occurred during the evolution of ancestral ruminants, gave rise to the three paralogous enzymes present in the bovine species. Herein we report data that demonstrate the existence of the orthologues of the bovine pancreatic, seminal, and cerebral ribonucleases coding sequences in the genomes of giraffe and sheep. The "seminal" sequence is a pseudogene in both species. We also report an analysis of the transcriptional expression of ribonuclease genes in sheep tissues. The data presented support a model for positive selection acting on the molecular evolution of ruminant ribonuclease genes.

Sequences related to the ox pancreatic ribonuclease coding region in the genomic DNA of mammalian species.

Mammalian pancreatic ribonucleases form a family of homologous proteins that has been extensively investigated. The primary structures of these enzymes were used to derive phylogenetic trees. These analyses indicate that the presence of three strictly homologous enzymes in the bovine species (the pancreatic, seminal, and cerebral ribonucleases) is due to gene duplication events which occurred during the evolution of ancestral ruminants. In this paper we present evidence that confirms this finding and that suggests an overall structural conservation of the putative ribonuclease genes in ruminant species. We could also demonstrate that the sequences related to ox ribonuclease coding regions present in genomic DNA of the giraffe species are the orthologues of the bovine genes encoding the three ribonucleases mentioned above.

Protein-DNA interactions in the 5'-flanking region of the bovine pancreatic ribonuclease gene.

In the 5'-flanking region of the bovine pancreatic ribonuclease gene a sequence has been identified which specifically binds one or more factors present in nuclear protein extracts prepared from bovine pancreas. The binding site, as delineated by footprinting analysis, is located in a region extending from positions -113 to -146 relative to the transcription initiation site of the ribonuclease gene. This region contains consensus sequences for known control transcriptional elements. The observed pattern of protein-DNA interactions is likely to be pancreas-specific as it could not be detected with nuclear extracts prepared from HeLa or bovine aorta endothelium cells.

A pancreatic-like ribonuclease is synthesized in rat brain.

The distribution and cell localization of a pancreatic-like ribonuclease (RNAase) in the rat brain has been studied by RNA blot analysis and in situ hybridization using as a probe the cDNA coding for the rat pancreas RNAase, and by immunocytochemistry using an antiserum raised against the rat pancreas RNAase. RNA blot analysis and in situ hybridization experiments have shown that the RNAase mRNA is present in all the cerebral areas investigated and that neurons appeared to be actively expressing RNAase mRNA while glial cells were devoid of hybridization signals. In agreement with these results the immunocytochemical analysis has shown that neurons are specifically immunostained. These experiments demonstrate that a pancreatic-like ribonuclease is synthesized in the neurons of the rat brain.

Molecular cloning of the gene encoding the bovine brain ribonuclease and its expression in different regions of the brain.

In this paper we report the molecular cloning of the gene encoding the bovine brain ribonuclease. The nucleotide sequence determined in this work shows a high degree of identity to the homologous gene encoding the bovine pancreatic ribonuclease. Processing of the primary transcripts of these genes also follows a similar pathway, splicing of the unique intron in the 5' untranslated region occurs at corresponding positions. Expression of the bovine brain ribonuclease gene can be detected both at the transcriptional and translational levels in all the regions of the brain examined.

Structure of the bovine pancreatic ribonuclease gene: the unique intervening sequence in the 5' untranslated region contains a promoter-like element.

Although pancreatic ribonucleases are extensively studied proteins, little information is available on nucleic acids coding for these enzymes. Here, for the first time, the structure of a gene coding for such an enzyme, the well known bovine pancreatic ribonuclease, is reported. The coding region of this gene is devoid of introns, whereas the 5' untranslated sequence of the pancreatic transcript contains an intron of 735 nucleotides. This intervening sequence is endowed with signals (CAAT and TATA boxes) which might act as regulatory elements. The structural organization of this gene suggests that the sequence coding for the bovine pancreatic ribonuclease might be expressed under the control of two different promoters.

Expression of opioid genes in bovine seminal vesicles.

In seminal vesicles, the organ producing most of seminal plasma in the bovine species, the pro-opiomelanocortin and the proenkephalin genes are transcribed and translated, and their translation products processed into opioid peptides, which are secreted into the seminal plasma. By using a micro-organ preparation of seminal vesicles we found that, after 20 h of incubation with labelled methionine, a multiplicity of opioids was produced. Among these, [Met]enkephalin and beta-endorphin were positively identified, whereas in the newly formed secretion only [Met]enkephalin was detected. This may be correlated to the finding that the concentration of beta-endorphin in an extract of seminal plasma was one order of magnitude lower than that of [Leu]enkephalin and [Met]enkephalin. These findings expand the picture of the presence of opioid peptides in the male reproductive tract, indicating that they should have a role(s) in the physiology of reproduction, not only in the hypothalamus-pituitary-gonadal axis, determining the reproductive potential, but also in the so-termed sex accessory glands, determining the actual events leading to reproduction. To our knowledge this is also the first case studied of opioid peptides produced as exocrine hormones.

Molecular evolution of pancreatic-type ribonucleases.

Amino acid sequences of 39 mammalian ribonucleases have been used to construct trees by the maximum parsimony procedure. These trees are in fairly good agreement with the biological classification of the species involved. In the branching order of the six investigated eutherian mammalian orders, the edentates diverge first, followed, probably, by the primates. No definite conclusions can be drawn about the order of divergence of the perissodactyls, the rodents, and the group consisting of artiodactyls plus cetaceans. Nucleic acid sequences of part of the messenger RNAs of rat pancreatic and bovine seminal ribonuclease were compared. Both messengers have a second stop codon at position 129, which is in agreement with the addition of four residues at the C-terminus in several other ribonucleases. Turtle pancreatic ribonuclease and human angiogenin differ from each other and from the mammalian ribonucleases at 55%-70% of the amino acid positions; they share a number of structural features. Mammalian nonsecretory ribonucleases are homologous to the pancreatic ribonucleases in sequence regions where the active-site histidine residues are located.

Sequence analysis of a cloned cDNA coding for bovine seminal ribonuclease.

The sequence of a cloned cDNA coding for bovine seminal ribonuclease, an enzyme secreted in the bull seminal vesicles, was determined. The cDNA starts at the amino acid residue 47 and terminates 12 nucleotides beyond the consensus sequence AAUAAA in the 3' non-coding region of the mRNA. Northern blotting analysis shows that the mRNA for bovine seminal ribonuclease consists of about 950 nucleotides, a value that is similar to that of other mRNAs coding for ribonucleases of the pancreatic type.

In vitro synthesis of pig pancreas ribonuclease.

From studies on the in vitro synthesis of the heavily glycosylated pig pancreas ribonuclease (molecular weight of the protein moiety is 13 786, on the basis of the amino acid composition), the following points emerge: (1) the enzyme is synthesized as a precursor having an apparent molecular weight about 7000 higher than that of the mature non-glycosylated protein; (2) the mRNA coding for the enzyme protein consists of about 950 nucleotides.

Proteins with preferential affinity for single-stranded DNA in tissues of Octopus vulgaris lam.

An investigation was carried out to search for proteins with preferential affinity for single-stranded DNA in nuclei of testicles, white bodies and optic lobes of Octopus Vulgaris Lam, as examples of organs characterized by high meiotic, high mitotic, and no or low mitotic activity, respectively. The results obtained are the following. Single strand binding proteins are present in testicles nuclei and, in much lower amount, in white bodies nuclei. Testicles cells have at least three protein species with affinity for single-stranded DNA, which, on the basis of elution characteristics and electrophoretic mobility, appear to be specific of testicle tissue. No single strand binding proteins could be found in Octopus optic lobes nuclei.

Influence of protein net charge on the nucleic acid helix-destabilizing activity of various pancreatic ribonucleases.

Helix-destabilization of double-stranded poly[d(A-T)]induced by various homologous pancreatic ribonucleases which differ in their net charges has been studied under different ionic strength conditions. The response of the destabilizing activity of the various proteins to ionic strength is represented by bell-shaped curves, whose maxima are shifted to higher ionic strength values the higher the number of positive charges of the RNAase involved in the nucleic acid-protein complex. This observation is discussed, and a model proposed, that could explain the experimental results presented.