Evolutionary history of introns in a multidomain globin gene

TheArtemia hemoglobin contains two sub-units that are similar or different chains of nine globin domains. The domains are ancestrally related and are presumed to be derived from copies of an original single-domain parent gene. Since the gene copies have remained in the same environment for several hundred million years they provide an excellent model for the investigation of intron stability. The cDNA for one of the two types of nine-domain subunit (domains T1–T9) has been sequenced. Comparison with the corresponding genomic DNA reveals a total of 17 intradomain introns. Fourteen of the introns are in locations on the protein that are conventional in globins of other species. In eight of the nine domains an intron corresponds to the B helix, amino acid B12, following the second nucleotide (phase 2), and in six domains a G-helix intron is located between G6 and G7 (phase 0). The consistency of this pattern is supportive of the introns having been inherited from a single-domain parent gene. The remaining three introns are in unconventional locations. Two occur in the F helix, either in amino acid F3 (phase 1) in domain T3, or between F2 and F3 (phase 0) in domain T6. The two F introns strengthen an interpretation of intron inheritance since globin F introns are rare, and in domains T3 and T6 they replace rather than supplement the conventional G introns, as though displacement from G to F occurred before that part of the gene became duplicated. It is inferred that one of the F introns subsequently moved by one nucleotide. Similarly, the third unconventional intron location is the G intron in domain T4 which is in G6, phase 2, one nucleotide earlier than the other G introns. Domain T4 is also unusual in lacking a B intron. The pattern of introns in theArtemia globin gene supports a concept of general positional stability but the exceptions, where introns have moved out of reading frame, or have moved by several codons, or have been deleted, suggest that intron displacements can occur after inheritance from an ancient source. Correspondence to: C.N.A. Trotman     

The sex-lethal gene homologue in Chrysomya rufifacies is highly conserved in sequence and exon-intron organization

A great variety of sex determination mechanisms exists in insect species. In Drosophila melanogaster sex is determined by the ratio between X chromosomes and autosomes, while in the blowfly Chrysomya rufifacies it is maternally determined. A cascade of genes which are involved in sex determination has been identified in D. melanogaster with the Sex-lethal gene (Sxl) as the key gene. We screened genomic libraries of C. rufifacies with a probe of the Sxl gene from D. melanogaster and isolated a genomic region that included most of the homologous gene. DNA- and protein-sequence comparison showed a high percent identity between the Chrysomya and the Drosophila gene. Up to 90% identity of the amino acid sequences was found in the region that contained the RNA-binding domains. The degree of identity is much lower outside of this functionally important region (18% identity). cDNA analysis showed a highly conserved exon-intron structure between the two species, although sex-specific splicing as used in D. melanogaster for the regulation of Sxl activity, could not be detected in C. rufifacies.     

Splicing Defects in the COL3A1 Gene: Marked Preference for 5′ (Donor) Splice-Site Mutations in Patients with Exon-Skipping Mutations and Ehlers-Danlos Syndrome Type IV

Ehlers-Danlos syndrome (EDS) type IV results from mutations in the COL3A1 gene, which encodes the constituent chains of type III procollagen. We have identified, in 33 unrelated individuals or families with EDS type IV, mutations that affect splicing, of which 30 are point mutations at splice junctions and 3 are small deletions that remove splice-junction sequences and partial exon sequences. Except for one point mutation at a donor site, which leads to partial intron inclusion, and a single base-pair substitution at an acceptor site, which gives rise to inclusion of the complete upstream intron into the mature mRNA, all mutations result in deletion of a single exon as the only splice alteration. Of the exon-skipping mutations that are due to single base substitutions, which we have identified in 28 separate individuals, only two affect the splice-acceptor site. The underrepresentation of splice acceptor-site mutations suggests that the favored consequence of 3' mutations is the use of an alternative acceptor site that creates a null allele with a premature-termination codon. The phenotypes of those mutations may differ, with respect to either their severity or their symptomatic range, from the usual presentation of EDS type IV and thus have been excluded from analysis.     

Isolation of the gene coding for Caenorhabditis elegans Rac2 homologue, a Ras-related small GTP-binding protein

When screening a Caenorhabditis elegans genomic library using the human Racl cDNA as probe, a hybridizing fragment of 2.7 kb was isolated which contained four exons with high sequence similarity to CeRacl, coding for the nematode homologue of the Ras-related small GTP-binding protein Racl. The putative translational product of 195 amino acids (aa) from the exons displayed 88% identity to the sequence of CeRacl. Interestingly, three alterations were found in the N-terminal ‘effector domain’ (residues 22–45) which hitherto was identical among all known Rac p21s, suggesting that CeRac2 might have different targets/functions for nematode development. Additionally, an insertion of 4 as was found in the hypervariable region at the C terminus of CeRac2.     

Validation of the Greater Hunter Native Vegetation Mapping as it pertains to the Upper Hunter region of New South Wales

Reliable vegetation maps are an important component of any long‐term landscape planning initiatives. A number of approaches are available but one, in particular, pattern recognition (segmentation) combined with modelling from floristic site data, is currently being used to map vegetation across NSW. An independent assessment of this approach based on a review of the Greater Hunter Native Vegetation Mapping (GHM_v4) was undertaken in order to assess its ability to cater for regional, local, strategic and landscape planning. The validation process tested 2151 locations across the Upper Hunter Valley region of New South Wales (NSW), Australia. The results suggest that mapping at the coarsest level of NSW vegetation classification, the Formation, is generally poor, with only Dry Sclerophyll Forest and Woodland modelled with some level of reliability. The modelled mapping of individual plant community types (PCTs) was found to be highly inaccurate with only 17% of validation points attributed as ‘correct’ and a further 13% ‘essentially correct’. Therefore, a majority of PCTs were mapped with an accuracy of less than 30%. The results of this validation suggest that the GHM_v4 is of such a low level of accuracy within the upper Hunter as to be inherently unusable for broad‐scale regional and local landscape planning or environmental assessment, including locating compensatory offsets for the loss of native vegetation due to developments. The GHM_v4 methods of pattern recognition of mainly SPOT5 satellite imagery combined with modelling from plot data have not produced reliable vegetation maps of plant community types. Yet this mapping programme is extending across NSW and could be misused for environmental decisions or as a regulation.     

Scapharca inæquivalvis Tetrameric Hemoglobin A and B Genes: Evidence for a Minigene

Vertebrate and many invertebrate globin genes have a three-exon/two-intron organization, with introns in highly conserved positions. According to the ``intron early' hypothesis, introns are the vestigial segments which flank previously independent coding sequences, thus providing evidence for the assembly of the ancient proteins by ``exon shuffling.' In this paper, we report the analysis of the genes of the bivalve mollusk Scapharca inaequivalvis tetrameric hemoglobin (HbII), which support this hypothesis, at least for the hemoglobin genes. We show the existence of ``minigenes' in the IIA and IIB globin genes, spanning part of the first and second introns, ``in frame' with the heme-binding domain coded by the second exon. Further support for the exon shuffling hypothesis can be found in the degree of identity of the ``new' translated sequences with those flanking the central protein domain of some invertebrate hemoglobins. Received: 31 July 1997 / Accepted: 12 December 1997     

Internodons as equivalence classes in genealogical networks: building-blocks for a rigorous species concept

Among the options suggested in phylogenetic systematics to solve the species problem is the Hennigian or internodal species concept. This concept interprets species as parts of the genealogical network of individual organisms between two successive permanent splits or between a permanent split and an extinction event. Though this option is at present not favoured by phylogeneticists, we believe that, to solve the species problem, there is no alternative to finding a satisfactory partition of the genealogical network. In previous work a formal definition has been developed of Hennigian or internodal species (called internodons here), based on a logical relation between individual organisms. In this paper, we prove that this definition indeed partitions genealogical networks exhaustively into mutually exclusive entities, by showing that the defining relation is an equivalence relation. Although internodons should not themselves be seen as species, they are essential building-blocks for any satisfying species concept.     

Stable tri-snRNP integration is accompanied by a major structural rearrangement of the spliceosome that is dependent on Prp8 interaction with the 5′ splice site

Exon definition is the predominant initial spliceosome assembly pathway in higher eukaryotes, but it remains much less well-characterized compared to the intron-defined assembly pathway. Addition in trans of an excess of 5′ss containing RNA to a splicing reaction converts a 37S exon-defined complex, formed on a single exon RNA substrate, into a 45S B-like spliceosomal complex with stably integrated U4/U6.U5 tri-snRNP. This 45S complex is compositonally and structurally highly similar to an intron-defined spliceosomal B complex. Stable tri-snRNP integration during B-like complex formation is accompanied by a major structural change as visualized by electron microscopy. The changes in structure and stability during transition from a 37S to 45S complex can be induced in affinity-purified cross-exon complexes by adding solely the 5′ss RNA oligonucleotide. This conformational change does not require the B-specific proteins, which are recruited during this stabilization process, or site-specific phosphorylation of hPrp31. Instead it is triggered by the interaction of U4/U6.U5 tri-snRNP components with the 5′ss sequence, most importantly between Prp8 and nucleotides at the exon–intron junction. These studies provide novel insights into the conversion of a cross-exon to cross-intron organized spliceosome and also shed light on the requirements for stable tri-snRNP integration during B complex formation.