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.     

Organization of non-vertebrate globin genes.

The organization of non-vertebrate globin genes exhibits substantially more variability than the three-exon, two-intron structure of the vertebrate globin genes. (1) The structures of genes of the single-domain globin chains of the annelid Lumbricus and the mollusc Anadara, and the globin gene coding for the two-domain chains of the clam Barbatia, are similar to the vertebrate plan. (2) Genes for single-domain chains exist in bacteria and protozoa. Although the globin gene is highly expressed in the bacterium Vitreoscilla, the putative globin gene hmp in E. coli, which codes for a chimeric protein whose N-terminal moiety of 139 residues contains 67 residues identical to the Vitreoscilla globin, may be either unexpressed or expressed at very low levels, despite the presence of normal regulatory sequences. The DNA sequence of the globin gene of the protozoan Paramecium, determined recently by Yamauchi and collaborators, appears to consist of two exons separated by a short intron. (3) Among the lower eukaryotes, the yeasts Saccharomyces and Candida have chimeric proteins consisting of N-terminal globin and C-terminal flavoprotein moieties of about the same size. The structure of the gene for the chimeric protein of Saccharomyces exhibits no introns. According to Riggs, the presence of chimeric proteins in E. coli and other prokaryotes, such as Alcaligenes and Rhizobium, as well as in yeasts, suggests a previously unrecognized evolutionary pathway for hemoglobin, namely that of a multipurpose heme-binding domain attached to a variety of unrelated proteins with diverse functions. (4) The published globin gene sequences of the insect larva Chironomus have an intron-less structure and are present as clusters of multiple copies; the expression of the globin genes is tissue and developmental stage-specific. Furthermore, the expression of many of these genes has not yet been demonstrated despite the presence of apparently normal regulatory sequences in the two flanking regions. Unexpectedly, Bergtrom and collaborators have recently shown that at least three Ctt globin II beta genes contain putative introns. (5) Pohajdak and collaborators have found a seven-exon and six-intron structure for the globin gene of the nematode Pseudoterranova which codes for a two-domain globin chain. Although the second and fourth introns of the N-terminal domain correspond to the two introns found in vertebrate globin genes, the position of the third intron is close to that of the central intron in plant hemoglobins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evolution of Orthologous Intronless and Intron-Bearing Globin Genes in Two Insect Species

While globin genes ctt-2β and ctt-9.1 in Chironomus thummi thummi each have a single intron, all of the other insect globin genes reported so far are intronless. We analyzed four globin genes linked to the two intron-bearing genes in C. th. thummi. Three have a single intron at the same position as ctt-2β and ctt-9.1; the fourth is intronless and lies between intron bearing genes. Finally, in addition to its intron, one gene (ctt-13RT) was recently interrupted by retrotransposition. Phylogenetic analyses show that the six genes in C. th. thummi share common ancestry with five globin genes in the distantly related species C. tentans, and that a 5-gene ancestral cluster predates the divergence of the two species. One gene in the ancestral cluster gave rise to ctn-ORFB in C. tentans, and duplicated in C. th. thummi to create ctt-11 and ctt-12. From parsimonious calculations of evolutionary distances since speciation, ctt-11, ctt-12, and ctn-ORFB evolved rapidly, while ctn-ORFE in C. tentans evolved slowly compared to other globin genes in the clusters. While these four globins are under selective pressure, we suggest that most chironomid globin genes were not selected for their unique function. Instead, we propose that high gene copy number itself was selected because conditions favored organisms that could synthesize more hemoglobin. High gene copy number selection to produce more of a useful product may be the basis of forming multigene families, all of whose members initially accumulate neutral substitutions while retaining essential function. Maintenance of a large family of globin genes not only ensured high levels of hemoglobin production, but may have facilitated the extensive divergence of chironomids into as many as 5000 species. Received: 31 December 1996 / Accepted: 16 May 1997     

Caenorhabditis Globin genes: Rapid intronic divergence contrasts with conservation of silent exonic sites

Globin genes from theCaenorhabditis speciesbriggsae andremanei were identified and compared with a previously describedC. elegans globin gene. The encoded globins share between 86% and 93% amino acid identity, with most of the changes in or just before the putative B helix.C. remanei was found to have two globin alleles,Crg1-1 andCrgl-2. The coding sequence for each is interrupted by a single intron in the same position. The exons of the two genes are only 1 % divergent at the nucleotide level and encode identical polypeptides. In contrast, intron sequence divergence is 16% and numerous insertions and deletions have significantly altered the size and content of both introns. Genetic crosses show thatCrg1-1 andCrgl-2 segregate as alleles. Homozygous lines for each allele were constructed and northern analysis confirmed the expression of both alleles. These data reveal an unusual situation wherein two alleles encoding identical proteins have diverged much more rapidly in their introns than the silent sites of their coding sequences, suggesting multiple gene conversion events. Correspondence to: D. Goldberg     

Scapharca inaequivalvis tetrameric hemoglobin A and B chains: cDNA sequencing and genomic organization

A and B globin cDNAs from the tetrameric hemoglobin of the bivalve molluscScapharca inaequivalvis were isolated by RT-PCR and sequenced. When compared with the biochemical data, the deduced protein sequences revealed only one amino acid substitution in the B chain. In order to investigate the genomic structure of these invertebrate globin genes, their intronic regions were amplified by PCR. The two genes showed the typical two-intron/three-exon organization found in vertebrates and seemed to reflect the ancestral gene structure, in accordance with the new globin gene evolution theory proposed by Dixon and Pohajadak (Trends Biochem. Sci. 17:486–488, 1992). The alternative hypothesis suggested by Go (Nature 291:90–92, 1981), that the central intron was lost during evolution, is also considered. In contrast to the related clamAnadara trapezia, S. inaequivalvis A and B globin genes were found to be present in multiple copies differing in intron size. In this study we report the complete sequences of the A (1,471 bp) and B (2,221 bp) globin genes, giving a detailed analysis of their intron features.     

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.     

A hemoglobin with an optical function

Hemoglobins are best known as oxygen transport proteins. Here we describe a hemoglobin from the parasitic nematode Mermis nigrescens (Mn-GLB-E) that has an optical, light shadowing function. The protein accumulates to high concentration as intracellular crystals in the ocellus of mature phototactic adult females while also being expressed at low concentration in other tissues. It differs in sequence and expression pattern from Mn-GLB-B, a second Mermis globin. It retains the structure and oxygen-binding and light-absorbing properties typical of nematode hemoglobins. As such, recruitment to a shadowing role in the eye appears to have occurred by changes in expression without modification of biochemistry. Both globins are coded by genes interrupted by two introns at the conserved positions B12.2 and G7.0, which is in agreement with the 3exon/2intron pattern model of globin gene evolution.     

Novel Repetitive Structures, Deviant Protein-Encoding Sequences andUnidentified ORFs in the Mitochondrial Genome of the BrachiopodLingula anatina

Complete sequence determination of the brachiopod Lingula anatina mtDNA (28,818 bp) revealed an organization that is remarkably atypical for an animal mt-genome. In addition to the usual set of 37 animal mitochondrial genes, which make up only 57% (16,555 bp) of the entire sequence, the genome contains lengthy unassigned sequences. All the genes are encoded in the same DNA strand, generally in a compact way, whereas the overall gene order is highly divergent in comparison with known animal mtDNA. Individual genes are generally longer and deviate considerably in sequence from their homologues in other animals. The genome contains two major repeat regions, in which 11 units of unassigned sequences and six genes (atp8, trnM, trnQ, trnV, and part of cox2 and nad2) are found in repetition, in the form of nested direct repeats of unparalleled complexity. One of the repeat regions contains unassigned repeat units dispersed among several unique sequences, novel repetitive structure for animal mtDNAs. Each of those unique sequences contains an open reading frame for a polypeptide between 80 and 357 amino acids long, potentially encoding a functional molecule, but none of them has been identified with known proteins. In both repeat regions, tRNA genes or tRNA gene-like sequences flank major repeated units, supporting the view that those structures play a role in the mitochondrial gene rearrangements. Although the intricate repeated organization of this genome can be explained by recurrent tandem duplications and subsequent deletions mediated by replication errors, other mechanisms, such as nonhomologous recombinations, appear to explain certain structures more easily.     

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.     

Conserved and variable repeat structures in the Balbiani ring gene family in Chironomus tentans

Summary The four Balbiani ring (BR) genes, BR1, BR2.1, BR2.2, and BR6 in the midge Chironomus tentans constitute a gene family encoding secretory proteins with molecular weights of approximately 10⁶ daltons. The major part of each gene is known to consist of tandemly organized composite repeat units resulting in a hierarchic repeat arrangement.Here, we present the sequence organization of the 5 part of the BR2.2 and BR6 genes and describe the entire transcribed part of the two genes. As the BR1 and BR2.1 genes were also fully characterized recently, this allows the comparison of all genes in the BR gene family.All four genes share the same exon-intron structure and have evolved by gene duplications starting from a common ancestor, having the same overall organization as the BR genes of today.The genes encode proteins that have an approximately 10,000-amino acid residue extended central domain, flanked by a highly charged, 200-residue amino-terminal domain and a globular 110-residue carboxy-terminal domain. Exons 1–3 and the beginning of exon 4 encode the amino-terminal domain, which throughout contains many regions built from short repeats. These repeats are often degenerate as to repeat unit and sequence and are present in different numbers between the genes. In several instances these repeat structures, however, are conserved at the protein level where they form positively or negatively charged regions.Each BR gene has a 26–38-kb-long exon 4, which consists of an array of 125–150 repeat units and encodes the central domain. The number of repeat units appears to be largely preserved by selection and all repeat units in the array are very efficiently homogenized. Occasionally variant repeats have been introduced, presumably from another BR gene by gene conversion, and spread within the array.Introns 1–3 at the 5 end of the genes have diverged extensively in sequence and length between the genes. In contrast, intron 4 at the 3 end is virtually identical between three of the four genes, suggesting that gene conversion homogenizes the 3 ends of the genes, but not the 5 ends. Offprint requests to: L. Wieslander     

Sequences and Evolution of Human and Squirrel Monkey Blue Opsin Genes

The sequences of the entire blue opsin gene in the squirrel monkey (Saimiri boliviensis) and the five introns of the human blue opsin gene were obtained. Intron 3 of these genes contains an Alu sequence and intron 4 contains a partial mer13 sequence. A comparison of the squirrel monkey opsin sequence with published mammalian opsin sequences shows that features believed to be functionally critical are all conserved. However, the blue opsin has evolved twice as fast as rhodopsin and is only as conservative as the β globin, which has evolved at the average rate of mammalian proteins. Interestingly, the interhelical loops are, on average, actually more conservative than the transmembrane α helical regions. The introns of the blue opsin gene have evolved at the average rate of introns in primate genes. Received: 5 August 1996 / Accepted: 2 October 1996     

Pheromone 4 gene of Euplotes octocarinatus

We have cloned and sequenced a 1.7 kb macronuclear chromosome encoding the pheromone 4 gene of Euplotes octocarinatus. The sequence of the secreted pheromone is preceded by a 42 amino acid leader peptide, which ends with a lysine residue. The sequence coding for the leader peptide contains information for a putative signal peptide and is interrupted by a 772 bp intron as shown by comparison with a cDNA clone. A 64 bp intron and a 145 bp intron interrupt the sequence coding for the secreted pheromone. The three introns contain typical 5′ and 3′ splice junctions and a putative branch point site. The small introns have a low GC content. The large intron has a GC content similar to that of the pheromone 4 gene exons. The amino acid sequence of pheromone 4, deduced from both the genomic DNA and the cDNA of pheromone 4, shows that the secreted pheromone consists of 85 amino acids. One of its amino acids is encoded by a UGA codon. Since it has been shown for pheromone 3 of E. octocarinatus that UGA is translated as cysteine, it is assumed that the UGA codon encodes cysteine in pheromone 4 as well. The 164 bp noncoding region upstream of the leader peptide is AT-rich and contains an inverted repeat capable of forming a stem-loop structure with a stem of 11 bp. The 151 bp noncoding region at the 3′ end of the chromosome contains a putative polyadenylation sequence and an inverted repeat. The macro-nuclear molecule is flanked by telomeres and carries the pentanucleotide motif TTGAA, located at a distance of 17 nucleotides from the telomeres. This motif has been suggested to be involved in the formation of macronuclear chromosomes. © 1992 Wiley-Liss, Inc.     

Nucleotide sequence of the goat embryonic alpha globin gene (zeta) and linkage and evolutionary analysis of the complete alpha globin cluster

In previous studies we identified and sequenced clones containing two adult alpha globin genes of the goat. Additional studies have revealed the presence of an embryonic alpha globin gene termed zeta. Sequence analysis of the gene shows that it is the largest mammalian or avian globin gene cloned to date. Its unusual size is mainly due to a 14 base-pair tandem repeat sequence in its first intron. A similar sequence is also found in the first intron of the human zeta gene. The goat zeta coding sequence differs greatly from that of the adult alpha, particularly at amino acid position 38, where it codes for the amino acid replacement of Gln for Thr. This change may confer a higher intrinsic O2 affinity on the zeta globin protein, ensuring a sufficient O2 supply for the developing goat embryo. The cloning and sequencing of this gene completes the alpha globin locus of the goat, composed of three genes in the following order 5'-zeta-I alpha-II alpha-3'. Evolutionary comparisons of the goat alpha locus with other amphibian, avian and mammalian loci reveal several interesting features. Statistical analysis confirms the hypothesis that the embryonic alpha gene is much older (400 million years) than the embryonic beta gene (200 million years), and that it is descended from a primordial gene, whose present-day counterpart is the Xenopus larval alpha globin gene. Our results also suggest that after the divergence of the avian line, the alpha A gene converted the alpha D gene during the evolution of the pre-mammalian line. The alpha D globin gene remains unconverted in the avian line, potentially because of insertion/deletion sequences that may prevent any gene conversion event. The divergence rates of specific globin genes have been analyzed and found to form an essentially straight line, in agreement with the neutralist view of evolution.     

The complete structure of the cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) chloroplast genome: Its composition and comparative analysis

The complete nucleotide sequence of the cucumber (C. sativus L. var. Borszczagowski) chloroplast genome has been determined. The genome is composed of 155,293 bp containing a pair of inverted repeats of 25,191 bp, which are separated by two single-copy regions, a small 18,222-bp one and a large 86,688-bp one. The chloroplast genome of cucumber contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species), and 37 tRNA genes (30 tRNA species), with 18 of them located in the inverted repeat region. Of these genes, 16 contain one intron, and two genes and one ycf contain 2 introns. Twenty-one small inversions that form stem-loop structures, ranging from 18 to 49 bp, have been identified. Eight of them show similarity to those of other species, while eight seem to be cucumber specific. Detailed comparisons of ycf2 and ycf15, and the overall structure to other chloroplast genomes were performed.     

Intergenic DNA sequences flanking the pseudo alpha globin genes of human and chimpanzee.

We have determined the sequence of 2400 base pairs upstream from the human pseudo alpha globin (psi alpha) gene, and for comparison, 1100 base pairs of DNA within and upstream from the chimpanzee psi alpha gene. The region upstream from the promoter of the psi alpha gene shows no significant homology to the intergenic regions of the adult alpha 2 and alpha 1 globin genes. The chimpanzee gene has a coding defect in common with the human psi alpha gene, showing that the product of this gene, if any, was inactivated before the divergence of human and chimpanzee. However the chimpanzee gene contains a normal ATG initiation codon in contrast to the human gene which has GTG as the initiation codon. The psi alpha genes of both human and chimpanzee are flanked by the same Alu family member. The structure and position of this repeat have not been altered since the divergence of human and chimpanzee, and it is at least as well conserved as its immediate flanking sequence. Comparing human and chimpanzee, the 300 bp Alu repeat has accumulated only two base substitutions and one length mutation; the adjacent 300 bp flanking region has accumulated five base substitutions and twelve length mutations.     

Rice chloroplast RNA polymerase genes: The absence of an intron in rpoC1 and the presence of an extra sequence in rpoC2

Summary The chloroplast genome contains sequences homologous to the Escherichia coli rpoA, rpoB and rpoC genes. The Choroplast rpoC gene is divided into rpoC1 and rpoC2, of which rpoC1 contains an intron. Comparison of the rice rpo genes with those from tobacco, spinach and liverwort revealed unique features of the rice genes; the lack of an intron in rpoC1 and the presence of an extra sequence of 381 by in rpoC2. The intron in rpoC1 is thus optional, and possible intron boundary sites in split rpoC1 genes can be estimated by comparison with rice rpoC1. The extra sequence is located in the middle of rpoC2 and has repeated structures. The amino acid sequence deduced from this sequence is extremely hydrophilic and anionic. The origin and function of this sequence are discussed.