Length variation and secondary structure of introns in the Mlc1 gene in six species of Drosophila

A nearly universal feature of intron sequences is that even closely related species exhibit a large number of insertion/deletion differences. The goal of the analysis described here is to test whether the observed pattern of insertion/deletion events in the genealogy of the myosin alkali light chain (Mlc1) gene is consistent with neutrality, and if not, to determine the underlying forces of evolutionary change. Mlc1 pre-mRNA is alternatively spliced, and one constraint is that signals necessary for tissue-specificity of directed splicing must be conserved. If the total length of an intron is functionally constrained, then the distribution of indels on branches of the gene genealogy should reflect a departure from randomness. Here we perform a phylogenetic analysis, inferring ancestral states wherever possible on a phylogeny of 29 alleles of Mlc1 from six species of Drosophila. Observed patterns of indels on the genealogy were compared to those from simulated data, with the result that we cannot reject the null hypothesis of neutrality. A clear departure from a neutral prediction was seen in the excess folding free energy predicted for the introns flanking the alternatively spliced exon. Relative rate tests also suggest a retardation in the rate of Mlc1 sequence evolution in the simulans clade.      

An ethidium bromide-agarose plate assay for the nonradioactive detection of cDNA synthesis

Ethidium bromide was used to determine the success of cDNA synthesis reactions. Since ethidium bromide in agarose can be used to quantitate RNA and DNA, conditions under which the greater fluorescence of double-stranded DNA (dsDNA) is utilized were devised to assay dsDNA synthesis from mRNA. Ethidium bromide at 5 micrograms/ml in agarose allowed quantitative detection of cDNA in the range of 0.03 to 0.0015 microgram. Sodium dodecyl sulfate had an adverse effect on the measurement of cDNA. Subsequent cDNA analysis by alkaline gel electrophoresis and staining in 5 micrograms/ml ethidium bromide allowed accurate and rapid sizing of cDNA and required only 0.1-0.05 microgram cDNA.     

Characterization of the Critical Amino Acids of an Aspergillus parasiticus Cytochrome P-450 Monooxygenase Encoded by ordA That Is Involved in the Biosynthesis of Aflatoxins B1, G1, B2, and G2

The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B₁, G₁, B₂, and G₂ requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B₁. Complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restored the ability to produce aflatoxins B₁, G₁, B₂, and G₂. The ordA gene placed under the control of the GAL1 promoter converted exogenously supplied OMST to aflatoxin B₁ in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the ordA1 gene had three point mutations which resulted in three amino acid changes (His-400→Leu-400, Ala-143→Ser-143, and Ile-528→Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the ordA gene in the synthesis of aflatoxins G₁, and G₂ in A. parasiticus suggests that enzymes required for the formation of aflatoxins G₁ and G₂ are not present in A. flavus. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. The ordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee.     

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.      

Sequence evolution and phylogenetic signal in control-region and cytochrome b sequences of rainbow fishes (Melanotaeniidae)

The nucleotide sequences of segments of the cytochrome b gene (351 bp), the tRNA(Pro) gene (49 bp), and the control region (approximately 313 bp) of mitochondrial DNA were obtained from 26 fish representing different populations and species of Melanotaenia and one species of Glossolepis, freshwater rainbow fishes confined to Australia and New Guinea. The purpose was to investigate relative rates and patterns of sequence evolution. Overall levels of divergence were similar for the cytochrome b and tRNA control-region sequences, both ranging from < 1% within subspecies to 15%-19% between genera. However, the patterns of sequence evolution differed. For the cytochrome b gene, transitions consistently exceeded transversions, the bias ranging from 4.2:1 to 2:1, depending on the level of sequence divergence. However, in the control-region sequence, a bias toward transitions (2:1) was observed only in comparisons between very similar sequences, and transversions outnumbered transitions in comparisons of divergent sequences. Graphic comparisons suggested that the control region was saturated for transitions at relatively low levels of sequence divergence but accumulated transversions at a greater rate than did the cytochrome b sequence. These distinct patterns of base substitution are associated with differences in A+T content, which is 70% for the tRNA control- region segment versus 50% for cytochrome b. A test for skewness in the distribution of lengths of random trees indicated that both segments contained phylogenetic signal. Parsimony analyses of the data from the two regions, with or without weighting schemes appropriate to the respective patterns of sequence evolution, identified the same five groupings of sequences, but the relationships among the groups differed. However, in most cases the branches uniting different combinations of groups were poorly supported, and the differences among topologies were insignificant. Considering the observed patterns of base substitution and the results of the phylogenetic analyses, we deduce that both the control region and cytochrome b are appropriate for population genetic studies but that the control region is less effective than cytochrome b for resolving relationships among divergent lineages of rainbow fishes.      

Crystal structure of human kynurenine aminotransferase II, a drug target for the treatment of schizophrenia

Kynurenic acid is an endogenous neuroactive compound whose unbalancing is involved in the pathogenesis and progression of several neurological diseases. Kynurenic acid synthesis in the human brain is sustained by the catalytic activity of two kynurenine aminotransferases, hKAT I and hKAT II. A wealth of pharmacological data highlight hKAT II as a sensible target for the treatment of neuropathological conditions characterized by a kynurenic acid excess, such as schizophrenia and cognitive impairment. We have solved the structure of human KAT II by means of the single-wavelength anomalous dispersion method at 2.3-A resolution. Although closely resembling the classical aminotransferase fold, the hKAT II architecture displays unique features. Structural comparison with a prototypical aspartate aminotransferase reveals a novel antiparallel strand-loop-strand motif that forms an unprecedented intersubunit beta-sheet in the functional hKAT II dimer. Moreover, the N-terminal regions of hKAT II and aspartate aminotransferase appear to have converged to highly similar although 2-fold symmetry-related conformations, which fulfill the same functional role. A detailed structural comparison of hKAT I and hKAT II reveals a larger and more aliphatic character to the active site of hKAT II due to the absence of the aromatic cage involved in ligand binding in hKAT I. The observed structural differences could be exploited for the rational design of highly selective hKAT II inhibitors.     

adhA in Aspergillus parasiticus Is Involved in Conversion of 5′-Hydroxyaverantin to Averufin

Two routes for the conversion of 5′-hydroxyaverantin (HAVN) to averufin (AVF) in the synthesis of aflatoxin have been proposed. One involves the dehydration of HAVN to the lactone averufanin (AVNN), which is then oxidized to AVF. Another requires dehydrogenation of HAVN to 5′-ketoaverantin, the open-chain form of AVF, which then cyclizes spontaneously to AVF. We isolated a gene, adhA, from the aflatoxin gene cluster of Aspergillus parasiticus SU-1. The deduced ADHA amino acid sequence contained two conserved motifs found in short-chain alcohol dehydrogenases—a glycine-rich loop (GXXXGXG) that is necessary for interaction with NAD⁺-NADP⁺, and the motif YXXXK, which is found at the active site. A. parasiticus SU-1, which produces aflatoxins, has two copies of adhA (adhA1), whereas A. parasiticus SRRC 2043, a strain that accumulates O-methylsterigmatocystin (OMST), has only one copy. Disruption of adhA in SRRC 2043 resulted in a strain that accumulates predominantly HAVN. This result suggests that ADHA is involved in the dehydrogenation of HAVN to AVF. Those adhA disruptants that still made small amounts of OMST also accumulated other metabolites, including AVNN, after prolonged culture.     

Pyrrolo[2,3-h]quinolinones: a new ring system with potent photoantiproliferative activity

A new class of compounds, the pyrrolo[2,3-h]quinolin-2-ones, nitrogen isosters of the angular furocoumarin Angelicin, was synthesized with the aim of obtaining new photochemotherapeutic agents with increased antiproliferative activity and lower undesired toxic effects than the lead compound. Two synthetic pathways were approached to allow the isolation both of the dihydroderivatives 10–17 and of the aromatic ring system 23. Compounds 10–17 showed a remarkable phototoxicity and a great UVA dose dependence reaching IC₅₀ values at submicromolar level. Intracellular localization of these compounds has been evaluated by means of fluorescence microscopy using tetramethylrhodamine methyl ester and acridine orange, which are specific fluorescent probes for mitochondria and lysosomes, respectively. A weak co-staining was observed with mitochondrial stain, whereas a specific localization in lysosomes was observed. Studies directed to elucidate the mode of action of this series of compounds revealed that they do not intercalate with DNA and do not induce photodamage to the macromolecule. On the contrary, they induce significative photodamage to lipids and proteins.     

Thiopyrano[2,3-e]indol-2-ones: angelicin heteroanalogues with potent photoantiproliferative activity

A new class of compounds, the thiopyrano[2,3-e]indol-2-ones, bioisosters of the angular furocoumarin angelicin, was synthesized with the aim of obtaining new photochemotherapeutic agents. In particular 7,8-dimethyl-thiopyranoindolone 6c s showed a remarkable phototoxicity and a great dose UVA dependence reaching IC(50) values at submicromolar level. This latter photoinduced a massive apoptosis and a remarkable photodamage to lipids and proteins. Although it did not intercalate DNA, it was able to cause photooxidation of DNA bases.