Cis- and trans-splicing and RNA editing are required for the expression of nad2 in wheat mitochondria

In wheat mitochondria, the gene coding for subunit 2 of the NADH-ubiquinone oxidoreductase (nad2) is divided into five exons located in two distant genomic regions. The first two exons of the gene, a and b, lie 22?kb downstream of exons c, d, and e, on the same DNA strand. All introns of nad2 are group II introns. A trans-splicing event is required to join exons b and c. It involves base pairing of the two precursor RNAs in the stem of domain IV of the intron. A gene coding for tRNA^Tyr is located upstream of exon c. In addition to splicing processes, mRNA editing is also required for the correct expression of nad2. The mature mRNA is edited at 36 positions, distributed over its five exons, resulting in 28 codon modifications. Editing increases protein hydrophobicity and conservation.     

Trans splicing in Oenothera mitochondria: nad1 mRNAs are edited in exon and trans-splicing group II intron sequences.

The complete NADH dehydrogenase subunit 1 (nad1) ORF in Oenothera mitochondria is encoded by five exons. These exons are located in three distant locations of the mitochondrial genome. One genomic region encodes exon a, the second encodes exons b and c, and the third specifies exons d and e. Cis-splicing group II introns separate exons b and c and d and e, while trans-splicing reactions are required to link exons a and b and c and d. The two parts of the group II intron sequences involved in these trans-splicing events can be aligned in domain IV. Exon sequences and the maturase-related ORF in intron d/e are edited by numerous C to U alterations in the mRNA. Two RNA editing events in the trans-splicing intron a/b improve conservation of the secondary structure in the stem of domain VI. RNA editing in intron sequences may thus be required for the trans-splicing reaction.     

A tripartite group II intron in mitochondria of an angiosperm plant

In mitochondria of flowering plants the nad5 open reading frame is assembled from five exons via two conventional cis-splicing and two trans-splicing events. Trans-splicing between exons c and d in wheat, petunia and Arabidopsis involves a bipartite group II intron structure, while in Oenothera a large portion of intron domains I–IV is missing from the major genomic locus. This intron region has been lost downstream of exon c and is now found in a distant genomic region. Intragenomic recombination across an 11 nucleotide sequence has separated these intron parts, which now have to be reassembled from three independent RNA precursors. This organisation coexists with highly substoichiometric copy numbers of the bipartite intron arrangement, consistent with an evolutionary origin of the tripartite intron by genomic disruption. Received: 28 August 1996 / Accepted: 11 December 1996     

Cloning and characterization of a third type of human α-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human α-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229–235; Tomita et al., Gene 76 (1989) 11–18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human α-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively.Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197–1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5′ region; hence the promoter lies far upstream relative to the other two AMY genes.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Interorganellar gene transfer in bryophytes: the functional nad7 gene is nuclear encoded in Marchantia polymorpha

The nad7 gene, encoding subunit 7 of NADH dehydrogenase, is mitochondrially encoded in seed plants. In the liverwort, Marchantia polymorpha, only a pseudogene is located in the mitochondrial genome. We have now identified the functional nad7 gene copy in the nuclear genome of Marchantia, coding for a polypeptide of 468 amino acids. The nuclear-encoded nad7 has lost the two group II introns present in the mitochondrial pseudogene copy. Instead, a typical nuclear intron is found to split an exon encoding the presumptive mitochondrial targeting signal peptide and the mature subunit 7 of NADH dehydrogenase. These results suggest that RNA-mediated gene transfer from the mitochondrial into the nuclear genome occurs not only in seed plants but also in bryophytes. Received: 11 March 1997 / Accepted: 20 August 1997     

Genomic organization of the human interleukin-12 receptor β2-chain gene

 The interleukin-12 receptor (IL-12R) is composed of two subunits, referred to as β1 and β2. Both chains are necessary for high-affinity IL-12 binding and signalling, although only the IL-12Rβ2 chain contains the intracellular tyrosine residues responsible for STAT4 activation. This study presents the intron-exon organization of the human IL-12Rβ2-chain gene. Polymerase chain reaction (PCR) primers designed across the cDNA (U46198) were used to trace introns, by comparing PCR product sizes obtained using cDNA and genomic DNA as templates. PCR products spanning introns were sequenced to determine the exact splice sites and flanking regions. The coding region of the gene was found to consist of 15 exons and 14 introns. All intron-exon boundaries are consistent with the consensus sequence for splice junctions (5′ GT/AG 3′). Comparison of the intron-exon organization with the human GCSFR gene indicated a remarkably well conserved genomic organization between these two class I cytokine receptors. Interestingly, we identified an alternatively spliced mRNA, encoding a putative, truncated protein, lacking all signalling potential. Received: 21 July 1999 / Revised: 2 September 1999     

Growth arrest of Schizosaccharomyces pombe following overexpression of mouse type 1 protein phosphatases

The rice disease resistance gene Xa21, which encodes a receptor-like kinase, is a member of a multigene family. Based on comparisons of genomic␣sequences of seven family members, seventeen transposon-like elements were identified in the 5′ and 3′ flanking regions and introns of these genes. Sequence characterization revealed that these elements are diverse, showing similarity to maize Ds, CACTA and miniature inverted repeat-like elements, as well as novel elements. Only two elements were located in presumed coding regions, indicating that integration of transposable elements at the Xa21 disease resistance locus occurred preferentially in noncoding regions. Received: 17 October 1997 / Accepted: 3 February 1998     

Deletion of the last two exons of the mitochondrialnad7 gene results in lack of the NAD7 polypeptide in aNicotiana sylvestris CMS mutant

InNicotiana sylvestris, two cytoplasmic male sterile (CMS) mutants obtained by protoplast culture show abnormal developmental features of both vegetative and reproductive organs, and mitochondrial gene reorganization following homologous recombination between 65 bp repeated sequences. A mitochondrial region of 16.2 kb deleted from both CMS mutants was found to contain the last two exons of thenad7 gene coding for a subunit of the mitochondrial respiratory chain complex I, which is encoded in the nucleus in fungi and animals but was recently found to be encoded by the mitochondrial genome in wheat. Although theN. sylvestris nad7 gene shows strong homology with its wheat counterpart, it contains only three introns instead of four. Polymerase chain reaction (PCR) experiments indicated that the parental gene organization, including the completenad7 gene, is probably maintained at a substoichiometric level in the CMS mutants, but this proportion is too low to have a significant physiological role, as confirmed by expression studies showing the lack of detectable amounts of the NAD7 polypeptide. Consequently, absence of NAD7 is not lethal to plant cells but a deficiency of complex I could be involved in the abnormal CMS phenotype.     

Genomic organization of a tumor growth inhibitor geneING1

ING1, a supposed tumor suppressor gene, codes for a p33 protein involved in cell proliferation control and regulation of apoptosis. A GenBank search revealed two groups of expressed sequence tags corresponding toING1 mRNA forms. The 3′ exon 2 is the same in both forms whereas the 5′ exons 1a and 1b differ.ING1-containing cosmids were found in the LA13NC05 library. EachING1 exon and flanking introns were sequenced using the cosmid 80H9 template. In the genome, the exons are arranged as 1b- 1a-2. RT-PCR showed that both mRNA forms are simultaneously present in cell lines. The deduced amino acid sequence for 1b-2 proved similar to those of human proteins ING1L (2e⁻⁷²) and ING1L-7 (6e⁻²⁴) and several proteins of lower eukaryotes having the ING-specific N-terminal domain and the zinc-binding domain PHD. Hence the ING-like proteins can be regarded as a separate evolutionarily old family. A peculiarity of theING1 structure is the CpG islands surrounding each of its three exons, suggesting regulation of its expression throughde novo methylation. The data on the fine structure ofING1 and its mRNA forms permit mutation screening and assessment of its methylation status in human tumor specimens.     

Extensive mis-splicing of a bi-partite plant mitochondrial group II intron

Expression of the seed plant mitochondrial nad5 gene involves two trans-splicing events that remove fragmented group II introns and join the small, central exon c to exons b and d. We show that in both monocot and eudicot plants, extensive mis-splicing of the bi-partite intron 2 takes place, resulting in the formation of aberrantly spliced products in which exon c is joined to various sites within exon b. These mis-spliced products accumulate to levels comparable to or greater than that of the correctly spliced mRNA. We suggest that mis-splicing may result from folding constraints imposed on intron 2 by base-pairing between exon a and a portion of the bi-partite intron 3 downstream of exon c. Consistent with this hypothesis, we find that mis-splicing does not occur in Oenothera mitochondria, where intron 3 is further fragmented such that the predicted base-pairing region is not covalently linked to exon c. Our findings suggest that intron fragmentation may lead to mis-splicing, which may be corrected by further intron fragmentation.     

Looking for Organization Patterns of Highly Expressed Genes: Purine-Pyrimidine Composition of Precursor mRNAs

We analyzed precursor messenger RNAs (pre-mRNAs) of 12 eukaryotic species. In each species, three groups of highly expressed genes, ribosomal proteins, heat shock proteins, and amino-acyl tRNA synthetases, were compared with a control group (randomly selected genes). The purine-pyrimidine (R-Y) composition of pre-mRNAs of the three targeted gene groups proved to differ significantly from the control. The exons of the three groups tested have higher purine contents and R-tract abundance and lower abundance of Y-tracts compared to the control (R-tract—tract of sequential purines with R _n ≥ 5; Y-tract—tract of sequential pyrimidines with Y _n ≥ 5). In species widely employing “intron definition” in the splicing process, the Y content of introns of the three targeted groups appeared to be higher compared to the control group. Furthermore, in all examined species, the introns of the targeted genes have a lower abundance of R-tracts compared to the control. We hypothesized that the R-Y composition of the targeted gene groups contributes to high rate and efficiency of both splicing and translation, in addition to the mRNA coding role. This is presumably achieved by (1) reducing the possibility of the formation of secondary structures in the mRNA, (2) using the R-tracts and R-biased sequences as exonic splicing enhancers, (3) lowering the amount of targets for pyrimidine tract binding protein in the exons, and (4) reducing the amount of target sequences for binding of serine/arginine-rich (SR) proteins in the introns, thereby allowing SR proteins to bind to proper (exonic) targets. (Reviewing Editor: Dr. Axel Meyer)     

Cotranscriptional expression of mitochondrial genes for subunits of NADH dehydrogenase, nad5, nad4, nad2, in Marchantia polymorpha

Three genes for the subunits of the NADH dehydrogenase (nad5, nad4, and nad2) are tandemly clustered on the liverwort mitochondrial genome. Their gene products showed high levels of amino acid sequence identity with the corresponding subunits from higher plant mitochondria (82.8–84.4%), and significant levels of identity with those from liverwort chloroplast (32.0–33.5%), Podospora anserina mitochondria (21.4–45.9%), and human mitochondria (18.4–27.9%). In addition, these three subunits from liverwort mitochondria have conserved amino acid residues in their central regions. The gene nad5 is interrupted by a 672 by group I intron, while genes nad4 and nad2 are interrupted by group II introns of 899 by and 1418 bp, respectively. Northern blot analysis using exon-intron specific probes indicated that these three genes are transcribed as a single precursor mRNA of 9.6 kb in length and are processed into mature mRNA molecules in liverwort mitochondria. Several regions of this nad gene cluster are repeated in the liverwort mitochondrial genome.Communicated by R.G. Herrmann     

The mitochondrial DNA of Dictyostelium discoideum: complete sequence, gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564 bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4 kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii. Received: 5 July 1999 / Accepted: 17 January 2000     

Molecular characterization of pdc2 and mapping of three pdc genes from rice

 The anaerobic fermentation pathway is thought to play an important role under flooding conditions. The pyruvate decarboxylase 2 (pdc2) gene that encodes the first enzyme of this pathway has been cloned and characterized from rice. This gene has an open reading frame that putatively encodes a 603 amino-acid-residue protein with a molecular mass of 64 kDa. pdc2 has five introns dispersed throughout the coding region, which is also true for rice pdc1. Although the length of these introns in rice pdc2 are different from those in rice pdc1, they are located in exactly the same positions based on the deduced amino-acid sequences. The temporal and spatial expression patterns of pdc1 and pdc2 show that pdc2 is induced to a higher level during the early period (1.5–12 h) of anoxia than pdc1, which is induced more after longer time periods (24–72 h) of anoxia in both shoots and roots. The map positions of the three pdc genes have also been determined. Rice pdc1 is located on chromosome 5 between BCD454A and RZ67, pdc2 is located on chromosome 3 between RZ329 and RZ313, and pdc3 is mapped on chromosome 7 distal to RG351. Received: 19 May 1998 / Accepted: 29 September 1998