Gene and antisense delivery in alcoholism research.

This article represents the proceedings of a symposium at the 2001 annual meeting of the Research Society on Alcoholism in Montreal, Canada. Drs. Yedy Israel and Fulton Crews were organizers and co-chairpersons. The presentations were (1) Introduction to the symposium, by Yedy Israel; (2) Gene delivery to the brain, by Fulton T. Crews; (3) Gene therapy in alcoholic liver injury, by Ronald Thurman; and (4) Antisense oligonucleotides and antisense-gene delivery, by Yedy Israel.

Structure and expression of a laccase gene from the ligninolytic basidiomycete Ceriporiopsis subvermispora.

A gene encoding laccase has been isolated from a genomic library of the white-rot basidiomycete Ceriporiopsis subvermispora constructed in Lambda GEM-11. This gene (Cs-lcs1) contains an open reading frame of 2215 bp, encoding a mature protein of 499 amino acids with a 21-residue signal peptide. The protein sequence exhibits between 63 and 68% identity with laccases from other basidiomycetes and shares with all of them 10 conserved histidines and one cysteine involved in the coordination of copper atoms at the active site of the enzyme. The gene possesses 11 introns, with splicing junctions and internal lariat formation sites adhering to the GT-AG and CTRAY rules, respectively. The upstream region of Cs-lcs1 contains a TATA box, two CAAT sites, five putative metal response elements and a ACE1 element. In agreement with the presence of the latter element, transcription of Cs-lcs1 is activated by copper and silver, as shown by Northern blot and reverse transcription followed by DNA amplification analyses. Based on Southern blot analysis, Cs-lcs1 appears to be the only gene encoding laccase in C. subvermispora.

Cloning and molecular analysis of a cDNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora.

A cDNA (MnP13-1) and the Cs-mnp1 gene encoding for an isoenzyme of manganese peroxidase (MnP) from C. subvermispora were isolated separately and sequenced. The cDNA, identified in a library constructed in the vector Lambda ZIPLOX, contains 1285 nucleotides, excluding the poly(A) tail, and has a 63% G+C content. The deduced protein sequence shows a high degree of identity with MnPs from other fungi. The mature protein contains 364 amino acids, which are preceded by a 24-amino-acid leader sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine and the distal arginine are conserved, although the aromatic binding site (L/V/I-P-X-P) is less hydrophilic than those of other peroxidases. A gene coding for the same protein (Cs-mnp1) was isolated from a genomic library constructed in Lambda GEM-11 vector using the cDNA MnP13-1 as a probe. A subcloned SacI fragment of 2.5kb contained the complete sequence of the Cs-mnp1 gene, including 162bp and 770bp of the upstream and downstream regions, respectively. The Cs-mnp1 gene possesses seven short intervening sequences. The intron splice junction sequences as well as the putative internal lariat formation sites adhere to the GT-AG and CTRAY rules, respectively. To examine the structure of the regulatory region of the Cs-mnp1 gene further, a fragment of 1.9kb was amplified using inverse PCR. A putative TATAA element was identified 5' of the translational start codon. Also, an inverted CCAAT element, SP-1 and AP-2 sites and several putative heat-shock and metal response elements were identified.

Lip-like genes in Phanerochaete sordida and Ceriporiopsis subvermispora, white rot fungi with no detectable lignin peroxidase activity.

Lignin peroxidase-like genes were PCR amplified from Phanerochaete sordida and Ceriporiopsis subvermispora, fungi lacking lignin peroxidase (LiP) activity. Amplification products were highly similar to previously described LiP genes. Using reverse transcription-coupled PCR a LiP-like cDNA clone was amplified from P. sordida RNA. In contrast, no evidence was obtained for transcription of C. subvermispora LiP genes.

Isolation and partial purification of mitochondrial and cytosolic rhodanese from liver of normal and p-dimethylaminoazobenzene treated mice.

Rhodanese (thiosulfate:cyanide sulfurtransferase, E.C. 2.8.1.1), an enzyme involved in heme regulation, showed distinctive mitochondrial and cytoplasmic activities in several models of tumorigenesis. To investigate the basis for these differences, the enzyme was partly purified and characterized from the mitochondrial and cytosolic liver fraction of mice treated with the carcinogen p-dimethyl-aminoazobenzene (DAB). A linear relationship between incubation time and specific activity was observed up to about 30 min for cytosolic enzyme and 15 min for mitochondrial enzyme irrespective of whether or not the enzyme was derived from treated or untreated animals. The optimum incubation temperature was 3 degrees C for the enzyme of both fractions in control animals and 30 degrees C for treated animals in both cases. In control and DAB treated animals the cytoplasmic rhodanese exhibited a maximum at a lower pH than for the mitochondrial enzyme. The enzyme showed typical Michaelis-Menten behavior with cyanide inhibition at concentrations higher than 25 mM for controls and 10 mM for treated animals for both fractions and thiosulfate inhibition at concentrations higher than 100 mM in all cases studied. Km values of 190 and 65.66 mM were obtained for thiosulfate and 6.37 and 9.79 mM for cyanide for both mitochondrial and cytosolic fractions of control animals; while Km values of 31.75 and 4.58 mM were obtained for thiosulfate and 0.61 and 1.11 mM for cyanide in both fractions of treated animals. We demonstrated differences in the kinetics for rhodanese derived from mitochondrial and cytoplasmic fractions of livers taken from tumor bearing mice. These differences might provide an explanation for the abnormalities of heme synthesis previously reported during hepatocarcinogenesis.