The exon/intron organization of the globin gene of Scapharca inaequivalvis homodimeric hemoglobin: unusual intron homology with other bivalve mollusc globin genes

In this study, we have investigated the positions of introns in the globin gene of Scapharca inaequivalvis homodimeric hemoglobin. We found the three exon/two intron organization typical of vertebrate globin genes, with the two introns in highly conserved positions, as it occurs in the A and B globin genes of the tetrameric hemoglobin from the same organism, confirming the absence of the so-called `central intron' found in the globin genes of plants and of some invertebrates. We identified two homodimeric globin genes (3207 and 2723 bp) that differ only with respect to the size of the first intron. Sequence analysis of the two first introns (1668 and 1364 bp) has revealed that they are highly homologous, except for a 569- and 296-bp insertion in each intron I. Interestingly, the two first introns contain regions with an unusually high identity (∼80%) with regions of the first intron of the congeneric clam Anadara trapezia and the related clam Barbatia reveana globin genes, suggesting that these uncoding regions may have played a regulatory role that has subsequently been lost during the course of the evolution.     

The hemoglobin gene of the deep-sea clam Calyptogena soyoae has a novel intron in A-helix

The cDNAs encoding two dimeric hemoglobins, Hbs I and II, of the deep-sea clam Calyptogena soyoae were amplified by PCR and the complete nucleotide sequences determined. The cDNA-derived amino acid sequences agreed completely with those determined chemically. Many of the molluscan intracellular globin genes have a characteristic four-exon/three-intron structure, with the precoding and two conventional introns conserved widely in animal globin genes. In this work we have determined the exon/intron organization of two hemoglobin genes of the deep-sea clam C. soyoae. Surprisingly, this gene has no precoding intron but instead contains an additional intron in the A-helix (A3.1), together with the two conventional introns (B12.2 and G6.3). This observation suggests that the precoding intron has been lost and the insertion of intron in A-helix occurred in the genes of Calyptogena. Alternatively, the sliding of intron from precoding to A-helix might have occurred.     

Gene Conversion and Evolution of Daphniid Hemoglobins (Crustacea, Cladocera)

The extracellular hemoglobins of cladocerans derive from the aggregation of 12 two-domain globin subunits that are apparently encoded by four genes. This study establishes that at least some of these genes occur as a tandem array in both Daphnia magna and Daphnia exilis. The genes share a uniform structure; a bridge intron separates two globin domains which each include three exons and two introns. Introns are small, averaging just 77 bp, but a longer sequence (2.2–3.2 kb) separates adjacent globin genes. A survey of structural diversity in globin genes from other daphniids revealed three independent cases of intron loss, but exon lengths were identical, excepting a 3-bp insertion in exon 5 of Simocephalus. Heterogeneity in the extent of nucleotide divergence was marked among exons, largely as a result of the pronounced diversification of the terminal exon. This variation reflected, in part, varying exposure to concerted evolution. Conversion events were frequent in exons 1–4 but were absent from exons 5 and 6. Because of this difference, the results of phylogenetic analyses were strongly affected by the sequences employed in this construction. Phylogenies based on total nucleotide divergence in exons 1–4 revealed affinities among all genes isolated from a single species, reflecting the impact of gene conversion events. In contrast, phylogenies based on total nucleotide divergence in exons 5 and 6 revealed affinities among orthologous genes from different taxa. Received: 8 March 1999 / Accepted: 14 July 1999     

Sequence, organization and expression of the core histone genes of Aspergillus nidulans

Summary The core histone gene family ofAspergillus nidulans was characterized. The H2A, H2B and H3 genes are unique in theA. nidulans genome. In contrast there are two H4 genes, H4.1 and H4.2. As previously reported for the H2A gene (May and Morris 1987) introns also interrupt the other core histone genes. The H2B gene, like the H2A gene, is interrupted by three introns, the H3 and H4.1 gene are each interrupted by two introns and the H4.2 gene contains one intron. The position of the single intron in H4.2 is the same as that the first intron of the H4.1 gene. The H2A and H2B genes are arranged as a gene pair separated by approximately 600 by and are divergently transcribed. The H3 and H4.1 genes are similarly arranged and are separated by approximately 800 bp. The H4.2 gene is not closely linked to either the H2A-H2B or H3-H4.1 gene pairs. Using pulse field gel electrophoresis an electrophoretic karyotype was established forA. nidulans. This karyotype was used to assign the H3–H4.1 gene pair and the H4.2 gene to linkage group VIII and the H2A–H2B gene pair to either linkage group III or VI. The abundance of each of the histone messenger RNAs was determined to be cell cycle regulated but the abundance of the H4.2 mRNA appears to be regulated differently from the others.     

Globin and linker sequences of the giant extracellular hemoglobin from the leech Macrobdella decora

A detailed electrospray ionization mass spectrometric study of the 3.5-MDa hexagonal bilayer hemoglobin (HBL Hb) from the pond leech Macrobdella decora has shown it to consist of at least six 17-kDa globin chains, of which two are monomeric and the remaining four occur as disulfide-bonded heterodimers, and three 24-kDa nonglobin linker chains (Weber et al., J. Mol. Biol. 251: 703–720, 1995). The cDNA sequences of the five major constituent chains, globin chains IIA, IIB, B, and C and linker chain L1, are reported here. The globins and linkers share 30%–50% and 20%–30% identity, respectively, with other annelid sequences. Furthermore, IIB and C align with strain A of annelid sequences, whereas IIA and B align with the strain B sequences. Although chains B and C are monomeric, chains IIA and IIB form the main disulfide-bonded dimer. They also have some unusual features: the distal His (E7) is replaced by Phe in IIA, and the highly conserved CD1Phe is replaced by Leu in IIB. In spite of these unusual features, the functional properties of Macrobdella Hb are comparable to those of other HBL Hbs. A phylogenetic analysis of the globin sequences from Macrobdella, the polychaete Tylorrhynchus, the oligochaete Lumbricus, and the vestimentiferan Lamellibrachia, indicates that the two strains originated by gene duplication followed by additional duplication of each of the two strains. The mutation rate of the linkers appeared to be faster than that of the globin chains. The phylogenetic trees constructed using the Maximum Likelihood, Neighbor-Joining and Fitch methods showed the Macrobdella globin sequences to be closest to Lumbricus, in agreement with a view of annelid evolution in which the divergence of the polychaetes occurred before the divergence of the leeches from oligochaetes.     

Phylogenetic and Exon–Intron Structure Analysis of Fungal Subtilisins: Support for a Mixed Model of Intron Evolution

Phylogenetic and exon–intron structure analyses of intra- and interspecific fungal subtilisins in this study provided support for a mixed model of intron evolution: a synthetic theory of introns-early and introns-late speculations. Intraspecifically, there were three phase zero introns in Pr1A and its introns 1 and 2 located at the highly conserved positions were phylogentically congruent with coding region, which is in favor of the view of introns-early speculation, while intron 3 had two different sizes and was evolutionarily incongruent with coding region, the evidence for introns-late speculation. Noticeably, the subtilisin Pr1J gene from different strains of M. ansiopliae contained different number of introns, the strong evidence in support of introns-late theory. Interspecifically, phylogenetic analysis of 60 retrievable fungal subtilisins provided a clear relationship between amino acid sequence and gene exon–intron structure that the homogeneous sequences usually have a similar exon–infron structure. There were 10 intron positions inserted by highly biased phase zero introns across examined fungal subtilisin genes, half of these positions were highly conserved, while the others were species-specific, appearing to be of recent origins due to intron insertion, in favor of the introns-late theory. High conservations of positions 1 and 2 inserted by the high percentage of phase zero introns as well as the evidence of phylogenetic congruence between the evolutionary histories of intron sequences and coding region suggested that the introns at these two positions were primordial.Reviewing Editor:Dr. Manyuan Long     

The sequence and phylogenesis of the α-globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon) and Cyprus mouflon (Ovis aries ophion)

In order to investigate the polymorphism of α-globin chain of hemoglobin amongst caprines, the linked ^Iα and ^IIα globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5′ and 3′ untranslated regions. European and Cyprus mouflons, which do not show polymorphic α globin chains, had almost identical α globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different α genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the ^Iα-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.     

Complete Structural Organization of the Human α1(V) Collagen Gene (COL5A1): Divergence from the Conserved Organization of Other Characterized Fibrillar Collagen Genes

Genes that encode the vertebrate fibrillar collagen types I–III have previously been shown to share a highly conserved intron/exon organization, thought to reflect common ancestry and evolutionary pressures at the protein level. We report here the complete intron/exon organization ofCOL5A1,the human gene that encodes the α1 chain of fibrillar collagen type V. The structure ofCOL5A1is shown to be considerably diverged from the conserved structure of the genes for fibrillar collagen types I–III.COL5A1has 66 exons, which is greater than the number of exons found in the genes for collagen types I–III. The increased number of exons is partly due to the increased size of the pro-α1(V) N-propeptide, relative to the sizes of the N-propeptides of the types I–III procollagen molecules. In addition, however, the increased number of exons is due to differences in the intron/exon organization of the triple-helix coding region ofCOL5A1compared to the organization of the triple-helix coding regions of the genes for collagen types I–III. Of particular interest is the increase of 54 bp exons in this region ofCOL5A1,strongly supporting the proposal that the triple-helix coding regions of fibrillar collagen genes evolved from duplication of a 54 bp primordial genetic element. Moreover, comparison of the structure ofCOL5A1to the highly conserved structure of the genes of collagen types I–III provides insights into the probable structure of the ancestral gene that gave rise to what appears to be two classes of vertebrate fibrillar collagen genes.     

Gene duplication and gene conversion in class II MHC genes of New Zealand robins (Petroicidae)

In contrast to mammals, the evolution of MHC genes in birds appears to be characterized by high rates of gene duplication and concerted evolution. To further our understanding of the evolution of passerine MHC genes, we have isolated class II B sequences from two species of New Zealand robins, the South Island robin (Petroica australis australis), and the endangered Chatham Island black robin (Petroica traversi). Using an RT-PCR based approach we isolated four transcribed class II B MHC sequences from the black robin, and eight sequences from the South Island robin. RFLP analysis indicated that all class II B loci were contained within a single linkage group. Analysis of 3-untranslated region sequences enabled putative orthologous loci to be identified in the two species, and indicated that multiple rounds of gene duplication have occurred within the MHC of New Zealand robins. The orthologous relationships are not retained within the coding region of the gene, instead the sequences group within species. A number of putative gene conversion events were identified across the length of our sequences that may account for this. Exon 2 sequences are highly diverse and appear to have diverged under balancing selection. It is also possible that gene conversion involving short stretches of sequence within exon 2 adds to this diversity. Our study is the first report of putative orthologous MHC loci in passerines, and provides further evidence for the importance of gene duplication and gene conversion in the evolution of the passerine MHC.Nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers AY258333–AY258335, AY428561–AY428570, and AY530534–AY530535     

A Comparison of Intraspecific Patterns of DNA Sequence Variation in Mitochondrial DNA, α-Enolase,and MHC Class II B Loci in Auklets (Charadriiformes: Alcidae)

Patterns of DNA sequence variation can be used to learn about mechanisms of organismal evolution, but only if mechanisms of sequence evolution are well understood. Although theories of molecular evolution are well developed, few empirical studies have addressed patterns and mechanisms of sequence evolution in nuclear genes within species. In the present study, we compared DNA sequences among three loci with different evolutionary constraints to determine the influences of effective population size, balancing selection, and linkage on intraspecific patterns of sequence variation. Specifically, we assessed the degree and nature of polymorphism in a 307-base pair (bp) fragment of the mitochondrial cytochrome b gene, intron VIII of the gene for -enolase (a presumably neutral nuclear gene), and an ~600-bp fragment of an MHC class II B gene, including 155 bp of the hypervariable peptide binding region (a nuclear locus thought to be under balancing selection) for least and crested auklets (Aethia pusilla and A. cristatella; Charadriiformes: Alcidae). Transspecies polymorphism was found in both -enolase and the MHC but not cytochrome b and, given estimates of effective population size, probably represents retained ancestral variation. Biases in nucleotide composition suggested that mutational bias, tRNA availability, and the secondary structure of mRNA and/or DNA may influence base usage. Several lines of evidence indicated that balancing selection may be acting on the MHC II B exon 2. However, no evidence of balancing selection was observed in the intron and exon sequences immediately downstream of MHC II B exon 2. Current address (Hollie E. Walsh): Department of Zoology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA     

Genomic Cloning and Characterization of a Novel Lectin Gene from <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis>

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.     

A nematode hemoglobin gene contains an intron previously thought to be unique to plants

Summary Hemoglobin genes from plants and animals both have a characteristic chromosomal organization. Plant hemoglobin genes contain a unique intron inserted into the heme-binding domain of exon 2. This intron has not been previously reported in animal globin genes, and its loss was hypothesized to have occurred early in the evolution of hemoglobins. We report here a unique six-intron, seven-exon internally duplicated nematode hemoglobin gene that contains an intron equivalent to the plant central intron in its first repeat. This nematode hemoglobin gene has lost both the central and the normal third intron in its second repeat. The nematode globin also contains a unique intron between its secretory peptide leader sequence and its coding sequence, which is absent in other extracellular invertebrate globin genes. Possible models to explain the head-to-tail duplication of this gene are discussed. Offprint requests to: B. Pohajdak     

Definitive RFLPs to distinguish between the human complement C4A/C4B isotypes and the major Rodgers/Chido determinants: Application to the study of C4 null alleles

Definitive restriction fragment length polymorphisms (RFLPs) representing the exact locations responsible for isotypicity between the human complement components C4A and C4B, and their generally associated major Rodgers (Rg1) and Chido (Ch1) antigenic determinants, have been designed. By means of a C4d-specific genomic probe for Southern blot analysis, a C4A gene can be defined by the presence of the 276 bp and 191 bp N 1 a IV fragments, while a C4B gene can be defined by a single 467 bp N1aIV fragment. In addition, an Rgl-expressing C4 gene can be represented by a 565 bp EcoO 109 fragment, and a Chl-expressing C4 gene by a 458 by EcoO 109 fragment, under the same conditions. All these polymorphic restriction fragments can be unambiguously and conveniently detected. In combination with the Taq I polymorphic patterns specific for the C4 loci and for the neighboring 21-hydroxylase genes, the nature and structure of the tandem C4,21-hydroxylase gene complex can be elucidated. In this study, it is inferred that the null allele of the HLA haplotype B44 DR6 C4A3 C4BQO is not a C4B allele, but probably encodes another C4A 3 allotype at the second C4 locus.Abbreviations used in this paper C4 (long) - C4 gene of 22 kb, with a 6–7 kb intron - C4 (short) - C4 gene of 16 kb, without a 6–7 kb intron; complotype SCO1, factor B S, C2 C, C4A QO; C4B 1 Dedicated to the memory of our teacher, the late Professor Rodney Porter C. H. F. R. S.     

The α-Globin Gene Family of an Australian Marsupial, Macropus eugenii: The Long Evolutionary History of the θ-Globin Gene and Its Functional Status in Mammals

Comparative evolutionary analyses of gene families among divergent lineages can provide information on the order and timing of major gene duplication events and evolution of gene function. Here we investigate the evolutionary history of the α-globin gene family in mammals by isolating and characterizing α-like globin genes from an Australian marsupial, the tammar wallaby, Macropus eugenii. Sequence and phylogenetic analyses indicate that the tammar α-globin family consists of at least four genes including a single adult-expressed gene (α), two embryonic/neonatally expressed genes (ζ and ζ′), and θ-globin, each orthologous to the respective α-, ζ-, and θ-globin genes of eutherian mammals. The results suggest that the θ-globin lineage arose by duplication of an ancestral adult α-globin gene and had already evolved an unusual promoter region, atypical of all known α-globin gene promoters, prior to the divergence of the marsupial and eutherian lineages. Evolutionary analyses, using a maximum likelihood approach, indicate that θ-globin, has evolved under strong selective constraints in both marsupials and the lineage leading to human θ-globin, suggesting a long-term functional status. Overall, our results indicate that at least a four-gene cluster consisting of three α-like and one β-like globin genes linked in the order 5′–ζ–α–θ–ω–3′ existed in the common ancestor of marsupials and eutherians. However, results are inconclusive as to whether the two tammar ζ-globin genes arose by duplication prior to the radiation of the marsupial and eutherian lineages, with maintenance of exon sequences by gene conversion, or more recently within marsupials.Reviewing Editor: Dr. John Oakeshott     

The cytochrome oxidase II gene in mitochondria of the sugar-beetBeta vulgaris L.

We have cloned and analyzed the sugar-beet mitochondrial gene for cytochrome oxidase subunit II (coxII). The sugar-beet and its deduced amino acid sequence were compared to its homologouscoxII gene sequences from both monocot and dicot plants. It was found to be highly conserved (89–95%) compared to homologue in other plant species. The 780 bp coding sequence of the sugar beetcoxII gene is interrupted at position 383 by a 1463 bp intron. This intron contains an additional 107 bp sequence that is not found in any of the plantcoxII genes studied thus far. The structure of the intron suggests that a large intron existed in an ancestralcoxII gene before monocots and dicots diverged in evolution. Three CGG codons in the sugar-beetcoxII coding sequence align with conserved tryptophan residues in the homologous gene of other species, suggesting that RNA editing takes place also in sugar-beet mitochondria. In 13 out of 24 codons ofcoxII mRNA that were found to be edited in four other plants, the sugar-beet gene already utilizes the edited codons. This phenomenon may indicate that the mitochondrial genome in sugar-beet is phylogenetically more archaic relative to these plants. An additional sequence of 279 bp that is identical to the first exon ofcoxII was identified in the mtDNA of the sugar-beet. This pseudo-gene is transcribed and its existence in the mitochondrial genome is unexplained.     

The evolution of protamine P1 genes in dasyurid marsupials

We report the complete DNA sequences of the protamine P1 gene and flanking regions for 13 species of the marsupial family Dasyuridae. The structure of the protamine locus is conserved in dasyurids and consists of two exons (of lengths 142–151 and 47 bp) separated by an intron (208–240 bp). A key feature of the dasyund intron is a 38–40 by duplication found in all species examined to date. This duplication apparently predates the radiation of modern dasyurid lineages and may be homologous to a similar feature in the marsupial mole (Notoryctes). Sequences from a species of Planigale demonstrate that this genus is unique among marsupials in possessing cysteine residues in its protamine P1 molecules. Cysteines may provide enhanced chemical stability for condensed sperm nuclei, a physiological feature that would converge on the common eutherian pattern. Phylogenetic analysis of the protamine genes yields a tree that is largely congruent with previous molecular systematic studies in two areas: (1) There are three main dasyurid lineages corresponding to the Sminthopsinae, Dasyurinae, and Phascogalinae; (2) Dasyurinae and Phascogalinae are sister groups. This study is the first estimate of dasyurid relationships based on a nuclear DNA sequence. Correspondence to: J.D. Retief     

Tandem duplication of alkaline phosphatase genes and polymorphism in the intergenic sequence in<Emphasis Type="Italic"> Bombyx mori</Emphasis>

Alkaline phosphatases are ubiquitous in organisms from bacteria to human. Two alkaline phosphatase genes, Alp-m and Alp-s, were independently cloned from the silkworm Bombyx mori. They were mapped to a small DNA region and shown to be organized in tandem. Exon-intron structures of the two genes were highly conserved, with the exception of the second intron in Alp-m, which has no counterpart in Alp-s. The similarity between the nucleotide sequences of the exons of the two genes was strikingly high (60–79%), suggesting that Alp-m and Alp-s originated from a duplication of their common ancestor gene. The intergenic sequence between the two Alp genes shows length polymorphism in different B. mori strains, which can be explained by presence/absence of two putative insertion sequences. This structural variation suggests a possible scenario for the divergence of the two Alp genes after the duplication event.Communicated by G. Reuter