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The apolipoprotein A-IV (ApoA-IV) plays a key role in lipid transport and feed intake regulation. In this work, four cDNA sequences encoding ApoA-IV paralogs were identified. Sequence analysis revealed conserved structural features including the common 33-codon block and nine repeated motifs. Gene structure analysis identified four exons and three introns except for apoA-IVAa1 (with only 3 exons). Synteny analysis showed that the four paralogs were structured into two clusters (cluster A containing apoA-IVAa1 and apoA-IVAa2 and cluster B with apoA-IVBa3 and apoA-IVBa4) linked to an apolipoprotein E. Phylogenetic analysis clearly separated the paralogs according to their cluster organization as well as revealed four subclades highly conserved in Acanthopterygii. Whole-mount analyses (WISH) in early larvae (0 and 1day post-hatch (dph)) showed that the four paralogs were mainly expressed in yolk syncytial layer surrounding the oil globules. Later, at 3 and 5dph, the four paralogs were mainly expressed in liver and intestine although with differences in their relative abundance and temporal expression patterns. Diet supply triggered the intensity of WISH signals in the intestine of the four paralogs. Quantification of mRNA abundance by qPCR using whole larvae only detected the induction by diet at 5dph. Moreover, transcript levels increased progressively with age except for apoA-IVAa2, which appeared as a low-expressed isoform. Expression analysis in juvenile tissues confirmed that the four paralogs were mainly expressed in liver and intestine and secondary in other tissues. The role of these ApoA-IV genes in lipid transport and the possible role of apoA-IVAa2 as a regulatory form are discussed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The mitochondrial genomes of Chlamydomonad algae lack the cox2 gene that encodes the essential subunit COX II of cytochrome c oxidase. COX II is normally a single polypeptide encoded by a single mitochondrial gene. In this work we cloned two nuclear genes encoding COX II from both Chlamydomonas reinhardtii and Polytomella sp. The cox2a gene encodes a protein, COX IIA, corresponding to the N-terminal portion of subunit II of cytochrome c oxidase, and the cox2b gene encodes COX IIB, corresponding to the C-terminal region. The cox2a and cox2b genes are located in the nucleus and are independently transcribed into mRNAs that are translated into separate polypeptides. These two proteins assemble with other cytochrome c oxidase subunits in the inner mitochondrial membrane to form the mature multi-subunit complex. We propose that during the evolution of the Chlorophyte algae, the cox2 gene was divided into two mitochondrial genes that were subsequently transferred to the nucleus. This event was evolutionarily distinct from the transfer of an intact cox2 gene to the nucleus in some members the Leguminosae plant family.

The algae of the family Chlamydomonadaceae lack the gene cox3 that encodes subunit III of cytochrome c oxidase in their mitochondrial genomes. This observation has raised the question of whether this subunit is present in cytochrome c oxidase or whether the corresponding gene is located in the nucleus. Cytochrome c oxidase was isolated from the colorless chlamydomonad Polytomella spp., and the existence of subunit III was established by immunoblotting analysis with an antibody directed against Saccharomyces cerevisiae subunit III. Based partly upon the N-terminal sequence of this subunit, oligodeoxynucleotides were designed and used for polymerase chain reaction amplification, and the resulting product was used to screen a cDNA library of Chlamydomonas reinhardtii. The complete sequences of the cox3 cDNAs from Polytomella spp. and C. reinhardtii are reported. Evidence is provided that the genes for cox3 are encoded by nuclear DNA, and the predicted polypeptides exhibit diminished physical constraints for import as compared with mitochondrial-DNA encoded homologs. This indicates that transfer of this gene to the nucleus occurred before Polytomella diverged from the photosynthetic Chlamydomonas lineage and that this transfer may have occurred in all chlamydomonad algae.

Recently, neural networks have been applied to a widening range of problems in molecular biology. An area particularly suited to neural-network methods is the identification of protein sorting signals and the prediction of their cleavage sites, as these functional units are encoded by local, linear sequences of amino acids rather than global 3D structures.

Most of the proteins that are used in mitochondria are imported through the double membrane of the organelle. The information that guides the protein to mitochondria is contained in its sequence and structure, although no direct evidence can be obtained. In this article, discriminant analysis has been performed with 47 parameters and a large set of mitochondrial proteins extracted from the SwissProt database. A computational method that facilitates the analysis and objective prediction of mitochondrially imported proteins has been developed. If only the amino acid sequence is considered, 75-97% of the mitochondrial proteins studied have been predicted to be imported into mitochondria. Moreover, the existence of mitochondrial-targeting sequences is predicted in 76-94% of the analyzed mitochondrial precursor proteins. As a practical application, the number of unknown yeast open reading frames that might be mitochondrial proteins has been predicted, which revealed that many of them are clustered.

The paper describes the Macintosh program MitoProt, which is suitable for studying mitochondrion-related proteins. MitoProt supplies a series of parameters that permit theoretical evaluation of mitochondrial targeting sequences, as well as calculation of the most hydrophobic fragment of 17 residues in the sequence, and a new parameter called mesohydrophobicity. The last two calculations are important for predicting the putative importability of a protein into mitochondria. Taken together, targeting sequence and hydrophobicity characteristics enable one to predict whether a given protein could be mitochondrial when no previous information on the nature of the sequence is available.

The apocytochrome b gene, exclusively encoded by the mitochondrial genome, was engineered so that it could be expressed in the yeast cytoplasm. Different combinations of the apocytochrome b transmembrane domains were produced in the form of hybrid proteins fused to both the N-terminal mitochondrial targeting sequence of the ATPase subunit 9 from Neurospora crassa and to a cytoplasmic version of the bI4 RNA maturase, localised on the N-terminal and C-terminal sides, respectively, of the hydrophobic stretches. The bI4 RNA maturase, which can complement mitochondrial mutations, was used as an in vivo reporter to assess the mitochondrial import of the different groups of transmembrane helices. This new, reliable and sensitive reporter activity allowed us to experimentally determine the limitations to the mitochondrial import of hydrophobic proteins. All eight transmembrane helices of apocytochrome b could be imported into mitochondria, either alone or in combination, but no more than three to four transmembrane helices could be imported together at one time. This limit is close to that observed in the population of nuclear-encoded mitochondrial proteins. The hydrophobic characteristics of engineered and natural proteins targeted to the mitochondrial inner membrane revealed two factors important in the import process. These were (a) the local hydrophobicity of a transmembrane segment, and (b) the average regional hydrophobicity of the protein over an extended length of 60-80 residues. Such features may have played a major role in the evolution of mitochondrial genomes.

A 2.1-kb DNA fragment containing the SWA2 gene determining an alpha-amylase from Schwanniomyces occidentalis has been sequenced. It contains an open reading frame of 1521 bp which has the potential to encode a 507 amino-acid protein of M(r) 55966. Its deduced amino-acid sequence shows significant similarities to the sequence of other studied alpha-amylases. These similarities identify a consensus sequence, F(LIV)(ED)NHD, which is shared in addition by most maltases, invertases and glucoamylases.

The SGA and STA2 genes that, respectively, encode the intra- and extracellular glucoamylases of Saccharomyces cerevisiae are coregulated negatively, at the level of transcription, by the STA10 gene. This finding was re-examined by determining the effects of STA10 on the expression of gene constructs containing different fragments from the SGA and STA2 promoter regions fused to the lacZ gene. Repression was observed only for promoter fragments carrying the sequence GTACAAG indicating that this element is responsible for the coregulation of SGA and STA2 by STA10.

In CYH2/cyh2 heterozygous diploids of the yeast Saccharomyces cerevisiae resistance is dominant over sensitivity at low (0.5-5 micrograms/ml) cycloheximide (cyh) concentrations. The cyh-resistant haploid strain MMY1 confers relatively high (10 micrograms/ml) cyh-resistance to heterozygous diploids constructed by mating this strain with cyh-sensitive haploid strains. We present here a genetic and biochemical study of strain MMY1. Analysis of tetrads obtained from a MMY1 heterozygous diploid showed that two unlinked nuclear mutations, determining high- and low-cycloheximide resistance, were present in MMY1. From a genomic library of this strain, constructed in vector YCp50, two plasmids (pRC1 and pRC13) have been isolated which, respectively, confer high- and low-resistance phenotypes to cyh-sensitive S. cerevisiae strains. The restriction maps of pRC1 and pRC13 are totally unrelated. This finding suggests that the genes harboring the two mutations encoding cyh-resistance from MMY1 were cloned in plasmids pRC1 and pRC13, respectively. Pulse field gel electrophoresis showed that the DNA insert of pRC1 maps at either chromosome VII or XV, whereas that from pRC13 maps at chromosome XI. This latter gene appears to define a previously unreported locus and has been named cyh5. By restriction and nucleotide sequencing analysis, the cyh gene present in pRC1 has been shown to correspond to cyh2, which maps at chromosome VII. These results suggest that the dominant cyh-resistance phenotype conferred by MMY1 in heterozygous diploids is promoted by the presence of both cyh2 and cyh5.(ABSTRACT TRUNCATED AT 250 WORDS)

The 5' region of the SGA and STA2 genes, encoding the intra- and extracellular glucoamylases, respectively, from Saccharomyces cerevisiae have been sequenced. In addition, the transcription initiation sites have been determined. Four distinct short elements (named I to IV) were found in both genes. Element III has the consensus sequence PuCATTTAPiG with a bilateral symmetry around the central T, and is present in both genes as a direct repeat. This motive seems responsible for the coregulation of STA2 and SGA by the repressor STA10 gene of S. cerevisiae.

A gene (SWA1) encoding an alpha-amylase activity from Schwanniomyces castellii has been cloned and expressed, via yeast cloning vector YEp13, in Saccharomyces cerevisiae. By using a riboprobe which is internal to the SWA1 gene, a 1.55 kb transcript was detected in the poly(A)+ RNA from both Sw. castellii and a S. cerevisiae clone harboring the SWA1 gene. This transcript should, therefore, correspond to the SWA1 gene. In addition, the DNA strand determining the alpha-amylase activity has been defined. Transcription of the SWA1 gene appears to be highly regulated in Sw. castellii, whereas it is constitutive in the S. cerevisiae harboring this gene.