The majority of long non-stop reading frames on the antisense strand can be explained by biased codon usage

In recent studies it has been suggested that long reading frames on the antisense strand of open reading frames (ORFs) are more frequent than expected. The vertebrate DNA database was searched for long (greater than 900 bp) antisense non-stop reading frames (aNRFs) that overlap known coding regions. The sequences obtained were predominantly positioned in DNA with a high usage of Gor C in the third codon position of the sense ORF. The major class of sequences revealed by the search was that of the heat-shock protein 70 kDa (Hsp70) family. A long Hsp70 aNRF was found in many Hsp70 sequences and occurred in species as diverse as fish, flies, fungi and bacteria. The role of codon usage bias was analysed both in the specific case of the Hsp70 genes and in a general species-wide context. The data obtained showed that even the very long aNRFs present in the Hsp70 family could be explained by codon usage bias on the sense strand. Codon usage bias is determined by GC content at the third codon position of the sense ORF and, in some species, by a high expression level of the gene in question. Such an explanation for the occurrence of long aNRFs cannot exclude that some aNRFs are transcribed and translated.     

Mitochondrial gene URFN of Neurospora crassa codes for a long polypeptide with highly repetitive structure

The mitochondrial DNA of Neurospora crassa contains a long potential gene, designated URFN, which is located immediately downstream from the CO1 gene. These two genes are encoded in different reading frames and overlap by 13 codons. URFN is 633 triplets long and terminates at a UAG stop codon. Its codon usage is atypical for N. crassa mitochondrial exons and introns, and resembles that of the long open reading frame (ORF) of the mitochondrial plasmid present in N. crassa strain Mauriceville. Multiple sequence repetitions occur in the presumptive URFN polypeptide, most notably a seven-times reiterated motif of 16 to 18 amino acid residues length. The hydropathy pattern shows that the N-terminal third of the URFN polypeptide is predominantly apolar and includes several potentially membrane-spanning stretches; the remaining part is hydrophilic. Calculation of the secondary structure predicts a high proportion (47%) of alpha-helix conformation. The longest alpha-helix contains 40 residues. No similarities to other mitochondrial genes or reading frames have been found, except a significant homology over a stretch of 16 amino acid residues between the N-terminal part of URFN and a well-conserved sequence in the C-terminal region of CO1. The repetitive region in URFN resembles a similarly repetitive stretch in an unassigned reading frame from bacteriophage lambda. Three arguments support the view that URFN is translated. The open reading frame has a considerable length; URFN is transcribed into a mRNA including the overlapping CO1 gene; URFN is most probably conserved among all the various Neurospora species examined thus far, strongly suggesting that it codes for an essential protein.     

Structure of a cyanobacterial gene encoding the 50S ribosomal protein L9

The rplI gene encoding the ribosomal protein L9 was found 4 kbp downstream from the desA gene, but on the opposite strand, in the genome of the cyanobacterium Synechocystis PCC6803. The deduced amino acid sequence is homologous to the sequences of the L9 proteins from Escherichia coli and chloroplasts of Arabidopsis and pea. The gene is present as a single copy in the chromosome and is transcribed as a mRNA of 0.64 kb. An open reading frame of unknown function (ORF291) was found in the upstream region of the rplI gene.     

A frame-specific symmetry of complementary strands of DNA suggests the existence of genes on the antisense strand

The bacterial DNA sequence in GenBank database were divided into coding and noncoding regions and examined for the base-trimer distribution in every triplet frame on the sense and antisense strands. The results revealed that for the noncoding region, both strands have very similar base-trimer distributions and have no frame specificity; that is, DNA is symmetric in the noncoding region. For the coding region, on the other hand, the symmetry is broken only in the triplet framework, and we found a special triplet-frame-specific symmetry which appears when the two complementary strands of the coding region are read from their 5 ends. In addition, the following frame specificity was also observed in the distribution of stop codons on the antisense strand of the coding region. When the antisense sequences of the open reading frames (ORFs) in the database are read in the three reading frames, the same reading frame as the corresponding ORF contains a significantly larger amount of long open frames without stop codons (i.e., nonstop frames [NSFs]) than expected, while the number of NSFs in the other two reading frames is similar to that of the expected one. That is, NSFs as well as ORFs are maintained in a frame-specific manner, and in this sense, DNA becomes symmetrical even in the coding region. These two kinds of frame-specific symmetries indicate that only an ORF and its complementary triplets are specifically recognized and maintained in DNA. We suppose that the antisense strands as well as the sense strands in the coding region may be transcribed, thereby producing various kinds of proteins corresponding to NSFs, though their amount may not be large. The presence of these proteins should have some benefits for living organisms, and therefore we propose that these proteins are upcoming enzymes having novel functions.Correspondence to: I. Urabe     

A group II intron in the Neurospora mitochondrial coI gene: nucleotide sequence and implications for splicing and molecular evolution.

The temperature-sensitive Neurospora nuclear mutant cyt18-1 is deficient in splicing many Group I mitochondrial introns when grown at its non-permissive temperature; however, splicing of intron 1 in the coI gene of the Adiopodoume (formerly called North Africa) strain is unaffected (R.A. Collins and A.M. Lambowitz, J. Mol. Biol. 184: 413-428, 1985). Here we show that coI intron 1 is a typical Group II intron, the only one identified to date in Neurospora. The differential effect of the cyt18-1 mutation suggests that splicing of certain introns could be regulated independently of others by nuclear-encoded proteins. The intron contains a long open reading frame (ORF) resembling that of the Neurospora Mauriceville mitochondrial plasmid. The intron and plasmid ORFs share unusual features of codon usage that suggest both evolved outside of the Neurospora mitochondrial genetic system.     

Natural antisense mRNAs to hyaluronan synthase 2 inhibit hyaluronan biosynthesis and cell proliferation

We report the identification of a natural antisense mRNA of hyaluronan synthase 2 that we have chosen to designate as HASNT (for HA synthase 2 antisense) in human and mouse. HASNT is transcribed from the opposite strand of the HAS2 gene locus and is represented by several independent expressed sequence tags in human. Portions of the mouse Hasnt gene were identified through an exon-trapping approach. Sequence conservation is extremely low between human and mouse HASNT, and it is not clear whether these mRNAs contain functional open reading frames. HASNT has an alternate splice site in both human and mouse. This splice site is located at an identical position within the gene in both species and results in mRNAs of two different lengths. In each species, the antisense portion of the HASNT gene is complementary to the first exon of HAS2, which represents the 5'-untranslated region. To study the biological activity of HASNT, two human expressed sequence tag clones, representing long and short HASNT splice variants, were cloned into a tetracycline-inducible vector and were stably transfected into human osteosarcoma U2-OS Tet-on cells. The long and short HASNT-expressing cells had a reduction in HAS2 mRNA levels up to 94 and 86%, respectively, whereas hyaluronan biosynthesis was inhibited by 40 and 37%, respectively. Cell proliferation was reduced throughout the time frame of the experiment. Exogenous high molecular mass hyaluronan failed to rescue the suppressed cell proliferation, whereas adenoviral-mediated overexpression of hyaluronan synthase 3, which stimulated endogenous hyaluronan biosynthesis, was able to rescue. Collectively, our data suggest that natural antisense mRNAs of HAS2 are able to regulate HAS2 mRNA levels and hyaluronan biosynthesis in a cell culture model system and may have an important and novel regulatory role in the control of HAS2, HA biosynthesis, and HA-dependent cell functions in vivo.     

Nucleotide sequence and intron structure of the apocytochrome b gene of Neurospora crassa mitochondria

The sequence of the apocytochrome b (cob) gene of Neurospora crassa has been determined. The structural gene is interrupted by two intervening sequences of approximately 1260 bp each. The polypeptide encoded by the exons shows extensive homology with the cob proteins of Aspergillus nidulans and Saccharomyces cerevisiae (79% and 60%, respectively). The two introns are, however, located at sites different from those of introns in the cob genes of A. nidulans and S. cerevisiae (which contain highly homologous introns at the same site within the gene). The introns share several short regions of sequence homology (10-12 bp long) with each other and with other fungal mitochondrial introns. Moreover, the second intron contains a 50 nucleotide long sequence that is highly homologous with sequences within every ribosomal intron of fungal mitochondria sequenced to date. The conserved sequences may allow the formation of a core secondary structure, which is nearly identical in many mitochondrial introns. The conserved secondary structure may be required for intron splicing. The second intron contains an open reading frame, continuous with the preceding exon, of approximately 290 codons. Two stretches of 10 amino acid residues, conserved in many introns, are present in the open reading frame.     

Duplication-induced mutation of a new Neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product.

A cloned Neurospora crassa genomic sequence, selected as preferentially transcribed when acetate was the sole carbon source, was introduced in extra copies at ectopic loci by transformation. Sexual crossing of transformants yielded acetate nonutilizing mutants with methylation and restriction site changes within both the ectopic DNA and the normally located gene. Such changes are typical of the duplication-induced premeiotic disruption (the RIP effect) first described by Selker et al. (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987). The mutants had the unusual phenotype of growth on ethanol but not on acetate as the carbon source. In a cross to the wild type of a mutant strain in which the original ectopic gene sequence had been removed by segregation, the acetate nonutilizing phenotype invariably segregated together with a RIP-induced EcoRI site at the normal locus. This mutant was transformed to the ability to use acetate by the cloned sequence. The locus of the mutation, designated acu-8, was mapped between trp-3 and un-15 on linkage group 2. The transcribed portion of the clone, identified by probing with cDNA, was sequenced, and a putative 525-codon open reading frame with two introns was identified. The codon usage was found to be strongly biased in a way typical of most Neurospora genes sequenced so far. The predicted amino acid sequence shows no significant resemblance to anything previously recorded. These results provide a first example of the use of the RIP effect to obtain a mutant phenotype for a gene previously known only as a transcribed wild-type DNA sequence.