Hypothetical Open Reading Frames Research Articles

Expressed sequence tag (EST)-based characterization of gene regulation in Artemia larvae

Summary Sequencing of cloned cDNAs to produce expressed sequence tag (EST) libraries is an efficient method for gene identification and analysis of transcription status within organisms. In this context, an EST library containing 609 sequences was constructed using cDNA from emerged larvae of the brine shrimp, Artemia franciscana, a developmental stage within this crustacean that has received limited attention at the molecular level. Three hundred and forty-eight ESTs exhibited similarity to database sequences of known function. Twenty sequences matched hypothetical open reading frames of unknown function, while 163 clones were unlike any archived database sequences and some of these may represent unidentified genes. Of the ESTs that encode proteins of known function, those participating in metabolic activities and protein/gene expression were the most numerous. Proteins involved in mitochondrial function and ribosome assembly were particularly prominent within these two functional categories. Other ESTs with multiple representatives in the library included those encoding actin, a selection of ion transporters, and proteins used in exoskeleton construction. Transcripts encoding proteins required for cell division were rare, suggesting that the number of dividing cells in emerged larvae is small. The data generated in this study significantly increase the scope of Artemia cDNA sequences in the public domain, and furnish a qualitative assessment of functional and biosynthetic activities within emerged Artemia larvae.

Identification, molecular cloning and expression of an α- N-acetylgalactosaminidase gene from Clostridium perfringens

The Clostridium perfringens gene encoding the previously characterized α- N-acetylgalactosaminidase (αNAG) was identified by protein microsequencing and database searching. The αNAG protein, designated AagA, was found to be encoded by a hypothetical gene of unknown function in the recently completed genome sequence of C. perfringens strain 13. The deduced translation product of 629 amino acid residues possessed a region of limited homology to several hypothetical open reading frames, an enterotoxin of unknown function and several known or predicted α-galactosidases, but did not exhibit homology to any of the multiple sequenced eukaryotic αNAG proteins. The C. perfringens aagA gene, encoding AagA, was cloned in an Escherichia coli T7 expression system, resulting in recombinants exhibiting high-level expression of the expected αNAG activity. To our knowledge, this is the first report of the cloning and expression of a bacterial αNAG-encoding gene and represents an important step in the development of recombinant αNAG as a tool in the enzymatic conversion of blood group antigens.

Identification, molecular cloning and expression of an alpha-N-acetylgalactosaminidase gene from Clostridium perfringens.

The Clostridium perfringens gene encoding the previously characterized alpha-N-acetylgalactosaminidase (alphaNAG) was identified by protein microsequencing and database searching. The alphaNAG protein, designated AagA, was found to be encoded by a hypothetical gene of unknown function in the recently completed genome sequence of C. perfringens strain 13. The deduced translation product of 629 amino acid residues possessed a region of limited homology to several hypothetical open reading frames, an enterotoxin of unknown function and several known or predicted alpha-galactosidases, but did not exhibit homology to any of the multiple sequenced eukaryotic alphaNAG proteins. The C. perfringens aagA gene, encoding AagA, was cloned in an Escherichia coli T7 expression system, resulting in recombinants exhibiting high-level expression of the expected alphaNAG activity. To our knowledge, this is the first report of the cloning and expression of a bacterial alphaNAG-encoding gene and represents an important step in the development of recombinant alphaNAG as a tool in the enzymatic conversion of blood group antigens.

Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae.

The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature alpha-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30 degrees C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process.

Distinguishing the ORFs from the ELFs: short bacterial genes and the annotation of genomes

A substantial fraction of hypothetical open reading frames (ORFs) in completely sequenced bacterial genomes are short, suggesting that many are not genes but random stretches of DNA. Although it is not feasible to authenticate the coding capacity of all such regions experimentally, comparisons of ORFs in related genomes can expose those that encode functional proteins.

Comparative proteome analysis of Helicobacter pylori.

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2-DE patterns with their identified protein species were added to the dynamic 2D-PAGE database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.

The ycf 9 (orf 62) gene in the plant chloroplast genome encodes a hydrophobic protein of stromal thylakoid membranes.

There are still some open reading frames, orfs, with unknown function in the higher plant chloroplast genome. Of these conserved orfs, designated as ycfs (hypothetical chloroplast open reading frames), one is ycf 9 (orf 62) in the transcription unit with the psbC and psbD genes. The aim of this work was to investigate the function of ycf 9 by insertional inactivation of the gene with a selectable marker cassette, consisting of the aadA coding region connected to the trc promoter and rrnB terminator. This cassette was inserted 19 bp downstream from the start of the coding region of the tobacco ycf 9 gene. Two DNA constructs with the aadA cassette in opposite orientations were precipitated on 1 micron gold particles and delivered into leaves of Nicotiana tabacum, cultivar Samsun, by the biolistic method. Spectinomycin-resistant plants regenerated following bombardment with only the construct containing the aadA gene in the opposite orientation as ycf 9. In spite of several subsequent regeneration cycles on spectinomycin, the transplastomic plants did not reach homoplasmicity. This suggests that the ycf 9 gene product is essential for chloroplast function. Using a polyclonal antibody raised against the inner part of the gene product, the polypeptide was localized in the stromal thylakoid membranes of chloroplasts.

Winter Flounder Expressed Sequence Tags: Establishment of an EST Database and Identification of Novel Fish Genes.

: An EST database of more than 900 sequences has been constructed from complementary DNAs from six different tissues (stomach, intestine, pyloric cecum, liver, spleen, and ovary) of the winter flounder Pleuronectes americanus. Template preparation and automated sequencing were optimized to generate high-quality information in an economic fashion. Using computer scripts developed in our laboratory, the sequences were automatically compared with sequences in the databases via a Web-browser interface, and significant returns were recorded and organized on user-friendly HTML pages. Half (453) of the ESTs had significant matches to database sequences of known function, 33 matched ESTs from other organisms, 34 matched ribosomal RNAs, and 24 matched hypothetical open reading frames of unknown function. Forty-one percent (374) of the ESTs had no matches to sequences in the database and presumably represent previously unidentified cDNAs. Several sequences are the first isolated from teleost fish, and should be of interest for gene mapping and studies of developmental biology.

The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae

The currently available yeast mitochondrial DNA (mtDNA) sequence is incomplete, contains many errors and is derived from several polymorphic strains. Here, we report that the mtDNA sequence of the strain used for nuclear genome sequencing assembles into a circular map of 85 779 bp which includes 10 kb of new sequence. We give a list of seven small hypothetical open reading frames (ORFs). Hot spots of point mutations are found in exons near the insertion sites of optional mobile group I intron-related sequences. Our data suggest that shuffling of mobile elements plays an important role in the remodelling of the yeast mitochondrial genome.