Sequential Loss of Two Neighboring Exons of the Tropoelastin Gene During Primate Evolution

Previous evidence has demonstrated the absence of exons 34 and 35 within the 3′ end of the human tropoelastin (ELN) gene. These exons encode conserved polypeptide domains within tropoelastin and are found in the ELN gene in vertebrate species ranging from chickens to rats to cows. We have analyzed the ELN gene in a variety of primate species to determine whether the absence of exons 34 and 35 in humans either is due to allelic variation within the human population or is a general characteristic of the Primates order. An analysis of the 3′ end of the ELN gene in several nonhuman primates and in 546 chromosomes from humans of varying ethnic background demonstrated a sequential loss of exons 34 and 35 during primate evolution. The loss of exon 35 occurred at least 35–45 million years ago, when Catarrhines diverged from Platyrrhines (New World monkeys). Exon 34 loss, in contrast, occurred only about 6–8 million years ago, when Homo separated from the common ancestor shared with chimpanzees and gorillas. Loss of both exons was probably facilitated by Alu-mediated recombination events and possibly conferred a functional evolutionary advantage in elastic tissue. Received: 6 July 1998 / Accepted: 18 February 1999     

Simulation of ‘hitch-hiking’ genealogies

An ancestral influence graph is derived, an analogue of the coalescent and a composite of Griffiths' (1991) two-locus ancestral graph and Krone and Neuhauser's (1997) ancestral selection graph. This generalizes their use of branching-coalescing random graphs so as to incorporate both selection and recombination into gene genealogies. Qualitative understanding of a ‘hitch-hiking’ effect on genealogies is pursued via diagrammatic representation of the genealogical process in a two-locus, two-allele haploid model. Extending the simulation technique of Griffiths and Tavaré (1996), computational estimation of expected times to the most recent common ancestor of samples of n genes under recombination and selection in two-locus, two-allele haploid and diploid models are presented. Such times are conditional on sample configuration. Monte Carlo simulations show that ‘hitch-hiking’ is a subtle effect that alters the conditional expected depth of the genealogy at the linked neutral locus depending on a mutation-selection-recombination balance. Received: 21 July 2000 / Published online: 5 December 2000     

Heterogeneity in Codon Usage in the Flatworm Schistosoma mansoni

Synonymous codon choices vary considerably among Schistosoma mansoni genes. Principal components analysis detects a single major trend among genes, which highly correlates with GC content in third codon positions and exons, but does not discriminate among putatively highly and lowly expressed genes. The effective number of codons used in each gene, and its distribution when plotted against GC₃, suggests that codon usage is shaped mainly by mutational biases. The GC content of exons, GC₃, 5′, 3′, and flanking (5′+ 3′+ introns) regions are all correlated among them, suggesting that variations in GC content may exist among different regions of the S. mansoni genome. We propose that this genome structure might be among the most important factors shaping codon usage in this species, although the action of selection on certain sequences cannot be excluded. Received: 10 March 1997 / Accepted: 27 June 1997     

Convergent evolution of major histocompatibility complex molecules in humans and New World monkeys

  In both Old World and New World monkeys Mhc-DRB sequences have been found which resemble human DRB1*03 and DRB3 genes in their second exon. The resemblance is shared sequence motifs and clustering of the genes or the encoded proteins in phylogenetic trees. This similarity could be due to common ancestry, convergence at the molecular level, or chance. To test which of these three explanations applies, we sequenced segments of New World monkey and macaque genes which encompass the entire second exon and large parts of both flanking introns. The test strongly supports the monophyly of New World monkey DRB intron sequences. The phylogenies of introns 1 and 2 from DRB1*03-like and DRB3-like genes are congruent, but both are incongruent with the exon 2-based phylogeny. The matching of intron 1- and intron 2-based phylogenies with each other suggests that reciprocal recombination has not played a major role in exon 2 evolution. Statistical comparisons of exon 2 from different DRB1*03 and DRB3 lineages indicate that it was neither gene conversion (descent), nor chance, but molecular convergence that has shaped their characteristic motifs. The demonstration of convergence in anthropoid Mhc-DRB genes has implications for the classification, age, and mechanism of generation of DRB allelic lineages. Received: 30 August 1999 / Revised: 19 October 1999     

Characterization of Mhc-DRB allelic diversity in white-tailed deer (Odocoileus virginianus) provides insight into Mhc-DRB allelic evolution within Cervidae

 Although white-tailed deer (Odocoileus virginianus) are one of North America's best studied mammals, no information is available concerning allelic diversity at any locus of the major histocompatibility complex in this taxon. Using the polymerase chain reaction, single-stranded conformation polymorphism analysis, and DNA sequencing techniques, 15 DRB exon 2 alleles were identified among 150 white-tailed deer from a single population in southeastern Oklahoma. These alleles represent a single locus and exhibit a high degree of nucleotide and amino acid polymorphism, with most amino acid variation occurring at positions forming the peptide binding sites. Furthermore, twenty-seven amino acid residues unique to white-tailed deer DRB alleles were detected, with 19 of these occurring at residues forming contact points of the peptide binding region. Significantly higher rates of nonsynonymous than synonymous substitutions were detected among these DRB alleles. In contrast to other studies of Artiodactyla DRB sequences, interallelic recombination does not appear to be playing a significant role in the generation of allelic diversity at this locus in white-tailed deer. To examine evolution of white-tailed deer (Odvi-DRB) alleles within Cervidae, we performed a phylogenetic analysis of all published red deer (Ceel-DRB), roe deer (Caca-DRB), and moose (Alal-DRB) DRB alleles. The phylogenetic tree clearly shows a trans-species persistence of DRB lineages among these taxa. Moreover, this phylogenetic tree provides insight into evolution of DRB allelic lineages within Cervidae and may aid in assignment of red deer DRB alleles to specific loci. Received: 25 June 1998 / Revised: 2 September 1998     

Genomic organization of the extracellular coding region of the human FGFR4 and FLT4 genes: Evolution of the genes encoding receptor tyrosine kinases with immunoglobulin-like domains

Receptor tyrosine kinases with five, seven, and three Ig-like domains in their extracellular region are grouped in subclasses IIIa, IIIb, and IIIc, respectively. Here, we describe the genomic organization of the extracellular coding region of the human FGFR4 (IIIc) and FLT4 (IIIb) genes and compare it to that of the human FGFR1(IIIc), KIT, and FMS (IIIa). The results show that while genes belonging to the same subclass have an identical exon/intron structure in their extracellular coding region—as they do in their intracellular coding region—genes of related subclasses only have a similar exon/intron structure. These results strongly support the hypothesis that the genes of the three subclasses evolved from a common ancestor by duplications involving entire genes, already in pieces. Hypotheses on the origin of introns and on the difference in the number of extracellular Ig-like domains in the three gene subclasses are discussed. Received: 19 August 1996 / Accepted: 2 January 1997     

Footprints of intragenic recombination at HLA loci

 To evaluate the effect of balancing selection and intragenic recombination (or gene conversion) at six individual HLA loci, synonymous nucleotide diversity in different exon groups is examined within (π_w) and between (π_b) allelic lineages that may be defined by either serological or DNA sequence differences. Both π values are high in exons which encode for the peptide binding region (PBR) and tend to decrease in other exons. The value of π_w is significantly smaller than that of π_b in any exon of any locus. However, even π_w is much greater than nucleotide diversity at non-HLA loci. These observations provide additional strong evidence for the operation of balancing selection in PBR-encoding exons and its indirect effects on polymorphism at linked neighboring regions. It appears that allelic lineages have generally evolved in isolation but the linkage relationships within and between exons are incomplete throughout the long evolutionary history. To quantify intragenic recombination and account for the large discrepancy between the HLA and non-HLA diversity, a population genetics model is analyzed with special reference to the evolution of modern humans. The analysis suggests that the recombination rate between two sites 1000 base pairs apart is about 10^–5 per generation and that the effective size of human populations (equivalent roughly to the number of breeding individuals in a randomly mating population) has dropped from 10⁵ to 10⁴ in most of the Quaternary. One possibility for this reduction is discussed. Received: 11 August 1997 / Revised: 8 October 1997     

Comparative Analysis of Evolution in a Rodent Histone H2a Pseudogene

Sequences were obtained from five species of rodents that are orthologous to an H2a histone pseudogene from Mus musculus. The pseudogene is part of the cluster of replication-dependent histone genes found on Mus musculus chromosome 13. Comparative analysis of these five sequences together with the previously published sequence from M. musculus shows that this gene has likely been a pseudogene throughout the evolution of the genus Mus, while the gene from Rattus norvegicus is likely functional. Three large (>20 bp) deletions were found among the Mus pseudogenes, a feature that is very unusual compared to surveys of processed pseudogenes. In addition, there are two single-base deletions and one 4-bp insertion among the Mus pseudogenes. The species distributions of one of the large deletions and the 4-bp insertion require either independent insertions of an identical sequence, independent deletions with identical boundaries, or a deletion followed by precise reintegration of the original sequence. The evidence favors the hypothesis of multiple deletions with identical boundaries. The ``coding' regions of the Mus pseudogenes show a much reduced level of among-species variability in the 3′ half of the pseudogene, compared both to the 5′ half and to flanking sequences. This supports a hypothesis that the 3′ end of the pseudogene is the target of frequent gene conversion by functional H2a genes. Received: 1 April 1997 / Accepted: 12 June 1997     

Intron Conservation in a UV-Specific DNA Repair Gene Encoded by Chlorella Viruses

Large dsDNA-containing chlorella viruses encode a pyrimidine dimer-specific glycosylase (PDG) that initiates repair of UV-induced pyrimidine dimers. The PDG enzyme is a homologue of the bacteriophage T4-encoded endonuclease V. The pdg gene was cloned and sequenced from 42 chlorella viruses isolated over a 12-year period from diverse geographic regions. Surprisingly, the pdg gene from 15 of these 42 viruses contain a 98-nucleotide intron that is 100% conserved among the viruses and another 4 viruses contain an 81-nucleotide intron, in the same position, that is nearly 100% identical (one virus differed by one base). In contrast, the nucleotides in the pdg coding regions (exons) from the intron-containing viruses are 84 to 100% identical. The introns in the pdg gene have 5′-AG/GTATGT and 3′-TTGCAG/AA splice site sequences which are characteristic of nuclear-located, spliceosomal processed pre-mRNA introns. The 100% identity of the 98-nucleotide intron sequence in the 15 viruses and the near-perfect identity of an 81-nucleotide intron sequence in another 4 viruses imply strong selective pressure to maintain the DNA sequence of the intron when it is in the pdg gene. However, the ability of intron-plus and intron-minus viruses to repair UV-damaged DNA in the dark was nearly identical. These findings contradict the widely accepted dogma that intron sequences are more variable than exon sequences. Received: 13 May 1999 / Accepted: 20 August 1999     

The Heat-Shock Gene hsp83 of Drosophila auraria: Genomic Organization, Nucleotide Sequence, and Long Antiparallel Coupled ORFs (LAC ORFs)

The genomic organization of the hsp83 gene of Drosophila auraria, a far-eastern endemic species belonging to the montium subgroup of the melanogaster species group, is presented here. Based on in situ hybridization on polytene chromosomes, cDNA and genomic clone mapping, nucleotide sequencing, and genomic Southern analysis, hsp83 is shown to be present as a single-copy gene at locus 64B on the 3L chromosome arm in D. auraria. This gene is organized into two exons separated by a 929-bp intron. The first exon represents the mRNA leader sequence and is not translated, while the coding region, having a length of 2,151 bp, is solely included in the second exon. Nucleotide sequence comparisons of D. auraria hsp83 with homologous sequences from other organisms show high conservation of the coding region (88–92% identity) in the genus Drosophila, in addition to the conserved genomic organization of two-exons–one-intron, of comparable size and arrangement. A phylogenetic tree based on the protein sequences of homologous genes from representative organisms is in accord with the accredited phylogenetic position of D. auraria. In the hsp83 gene region, a second case of long antiparallel coupled open reading frames (LAC ORFs) for this species was found. The antiparallel to the hsp83 gene ORF is 1,554 bases long, while the two ORFs overlap has a size of 1,548 bp. The anti-hsp83 ORF does not show significant homology to any known gene sequences. In addition, no similar LAC ORF structures were found in homologous gene regions of other organisms. Received: 18 April 1997 / Accepted: 1 August 1997     

A Complex Organization of the Gene Encoding Cytochrome Oxidase Subunit 1 in the Mitochondrial Genome of the Dinoflagellate, Crypthecodinium cohnii: Homologous Recombination Generates Two Different cox1 Open Reading Frames

In the course of investigating mitochondrial genome organization in Crypthecodinium cohnii, a non-photosynthetic dinoflagellate, we identified four EcoRI fragments that hybridize to a probe specific for cox1, the gene that encodes subunit 1 of cytochrome oxidase. Cloning and sequence characterization of the four fragments (5.7, 5.1, 4.1, 3.5 kilobase pairs) revealed that cox1 exists in four distinct but related contexts in C. cohnii mtDNA, with a central repeat unit flanked by one of two possible upstream (flanking domain 1 or 2) and downstream (flanking domain 3 or 4) regions. The majority of the cox1 gene is located within the central repeat; however, the C-terminal portion of the open reading frame extends into flanking domains 3 and 4, thereby creating two distinct cox1 coding sequences. The 3′-terminal region of one of the cox1 reading frames can assume an elaborate secondary structure, which potentially could act to stabilize the mature mRNA against nucleolytic degradation. In addition, a high density of small inverted repeats (15–22 base pairs) has been identified at the 5′-end of cox1, further suggesting that hairpin structures could be important for gene regulation. The organization of cox1 in C. cohnii mtDNA appears to reflect homologous recombination events within the central repeat between different cox1 sequence contexts. Such recombining repeats are a characteristic feature of plant (angiosperm) mtDNA, but they have not previously been described in the mitochondrial genomes of protists. Received: 21 December 2000 / Accepted: 30 January 2001     

Partial Sequence of a Sponge Mitochondrial Genome Reveals Sequence Similarity to Cnidaria in Cytochrome Oxidase Subunit II and the Large Ribosomal RNA Subunit

A 2550-bp portion of the mitochondrial genome of a Demosponge, genus Tetilla, was amplified from whole genomic DNA extract and sequenced. The sequence was found to code for the 3′ end of the 16S rRNA gene, cytochrome c oxidase subunit II, a lysine tRNA, ATPase subunit 8, and a 5′ portion of ATPase subunit 6. The Porifera cluster distinctly within the eumetazoan radiation, as a sister group to the Cnidaria. Also, the mitochondrial genetic code of this sponge is likely identical to that found in the Cnidaria. Both the full COII DNA and protein sequences and a portion of the 16S rRNA gene were found to possess a striking similarity to published Cnidarian mtDNA sequences, allying the Porifera more closely to the Cnidaria than to any other metazoan phylum. The gene arrangement, COII—tRNA^Lys—ATP8—ATP6, is observed in many Eumetazoan phyla and is apparently ancestral in the metazoa. Received: 24 November 1997 / Accepted: 14 September 1998     

Frequent gene conversion between human red and green opsin genes

To study the evolution of human X-linked red and green opsin genes, genomic sequences in large regions of the two genes were compared. The divergences in introns 3, 4, and 5 and the 3′ flanking sequence of the two genes are significantly lower than those in exons 4 and 5. The homogenization mechanism of introns and the 3′ flanking sequence of human red and green opsin genes is probably gene conversion, which also occurred in exons 1 and 6. At least one gene conversion event occurred in each of three regions (1, 3, and 5) in the sequences compared. In conclusion, gene conversion has occurred frequently between human red and green opsin genes, but exons 2, 3, 4, and 5 have been maintained distinct between the two genes by natural selection. Received: 29 September 1997 / Accepted: 29 September 1997     

Mitochondrial DNA of the coral sarcophyton glaucum contains a gene for a homologue of bacterial muts: A possible case of gene transfer from the nucleus to the mitochondrion

The nucleotide sequences of two segments of 6,737 ntp and 258 ntp of the 18.4-kb circular mitochondrial (mt) DNA molecule of the soft coral Sarcophyton glaucum (phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea) have been determined. The larger segment contains the 3′ 191 ntp of the gene for subunit 1 of the respiratory chain NADH dehydrogenase (ND1), complete genes for cytochrome b (Cyt b), ND6, ND3, ND4L, and a bacterial MutS homologue (MSH), and the 5′ terminal 1,124 ntp of the gene for the large subunit rRNA (l-rRNA). These genes are arranged in the order given and all are transcribed from the same strand of the molecule. The smaller segment contains the 3′ terminal 134 ntp of the ND4 gene and a complete tRNA^f-Met gene, and these genes are transcribed in opposite directions. As in the hexacorallian anthozoan, Metridium senile, the mt-genetic code of S. glaucum is near standard: that is, in contrast to the situation in mt-genetic codes of other invertebrate phyla, AGA and AGG specify arginine, and ATA specifies isoleucine. However, as appears to be universal for metazoan mt-genetic codes, TGA specifies tryptophan rather than termination. Also, as in M. senile the mt-tRNA^f-Met gene has primary and secondary structural features resembling those of Escherichia coli initiator tRNA, including standard dihydrouridine and TψC loop sequences, and a mismatched nucleotide pair at the top of the amino-acyl stem. The presence of a mutS gene homologue, which has not been reported to occur in any other known mtDNA, suggests that there is mismatch repair activity in S. glaucum mitochondria. In support of this, phylogenetic analysis of MutS family protein sequences indicates that the S. glaucum mtMSH protein is more closely related to the nuclear DNA-encoded mitochondrial mismatch repair protein (MSH1) of the yeast Saccharomyces cerevisiae than to eukaryotic homologues involved in nuclear function, or to bacterial homologues. Regarding the possible origin of the S. glaucum mtMSH gene, the phylogenetic analysis results, together with comparative base composition considerations, and the absence of an MSH gene in any other known mtDNA best support the hypothesis that S. glaucum mtDNA acquired the mtMSH gene from nuclear DNA early in the evolution of octocorals. The presence of mismatch repair activity in S. glaucum mitochondria might be expected to influence the rate of evolution of this organism's mtDNA. Received: 13 January 1997 / Accepted: 23 September 1997     

Homologous recombination involving cox2 is responsible for a mutation in the CMS-specific mitochondrial locus of Petunia

We have characterized the only mutation detected so far in S-Pcf, the mitochondrial cytoplasmic male sterility (CMS)-specific locus of petunia. This locus consists of three open reading frames (ORFs): the first contains part of atp9, an intron-less cox2 pseudogene (which does not contain the original cox2 ATG) and the unidentified reading frame urf-s; the second and third ORFs correspond to the only copies of nad3 and rps12 genes in the genome, respectively. In the cell line R13-138, which was generated from a male-sterile somatic hybrid (line SH13-138), a change in the first ORF of the S-Pcf locus has been characterized: the atp9 sequence has been lost, while exon1 of the normal copy of the cox2 gene (including the original ATG sequence) and the adjacent 5′ sequence of the petunia recombination repeat, have been introduced. The data suggest that this reorganization of mtDNA is the consequence of a homologous recombination event involving part of the cox2 coding region, and that the cox2 coding region may serve as an active site for inter- or intra-mtDNA homologous recombination. The results further suggest that in line SH13-138 (or during its maintenance in tissue culture), segregation of the S-Pcf-containing mtDNA molecules has occurred, and the mutant mtDNA is now predominant in the population. Received: 9 September 1996 / Accepted: 27 January 1997     

Activation of the Cardiac Ryanodine Receptor by Sulfhydryl Oxidation is Modified by Mg2+ and ATP

The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca²⁺] of 10⁻⁷ m (in the absence of Mg²⁺ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm cis Mg²⁺ (10⁻⁷ m cis Ca²⁺) or by 10 mm cis Mg²⁺ (10⁻³ m cis Ca²⁺), or activated by 4 mm ATP (10⁻⁷ m cis Ca²⁺), also responded to 1 mm cis 4,4′-DTDP with activation and then loss of activity. P _o and mean open time (T _o ) of the maximally activated channels were lower in the presence of Mg²⁺ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg²⁺, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca²⁺ or caffeine (Eager et al., 1997), the activation produced by 1 mm cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation. Received: 8 September 1997/Revised: 20 January 1998     

Preference for Internucleotide Linkages as a Function of the Number of Constituents in a Mixture

Phosphoimidazolide-activated ribomononucleotides (*pN; see Scheme I) are useful substrates for the nonenzymatic synthesis of oligonucleotides. In the presence of metal ions dilute neutral aqueous solutions of *pN (0.01 M) typically yield only small amounts of dimers and traces of oligomers; most of *pN hydrolyzes to yield nucleoside 5′-monophosphate (5′NMP). An earlier investigation of *pN reactions in highly concentrated aqueous solutions (up to 1.4 M) showed, as expected, that the percentage yield of the condensation products increases and the yield of the hydrolysis product correspondingly decreases with *pN concentration (Kanavarioti 1997). Here we report product distributions in reactions with one, two, or three reactive components at the same total nucleotide concentration. *pN used as substrates were the nucleoside 5′-phosphate 2-methylimidazolides, 2-MeImpN, with N= cytidine (C), uridine (U), or guanosine (G). Reactions were conducted as self-condensations, i.e., one nucleotide only, with two components in the three binary U,C, U,G, and C,G mixtures, and with three components in the ternary U,C,G mixture. The products are 5′NMP, 5′,5′-pyrophosphate-, 2′,5′-, 3′,5′-linked dimers, cyclic dimers, and a small percentage of longer oligomers. The surprising finding was that, under identical conditions, including the same total monomer concentration, the product distribution differs substantially from one reaction to another, most likely due to changing intermolecular interactions depending on the constituents. Even more unexpected was the observed trend according to which reactions of the U,C,G mixture produce the highest yield of internucleotide-linked dimers, whereas the self-condensations produce the least and the reactions with the binary mixtures produce yields that fall in between. What is remarkable is that the approximately two-fold increase in the percentage yield of internucleotide-linked dimers is not due to a concentration effect or a catalyst, but to the increased complexity of the system from a single to two and three components. These observations, perhaps, provide an example of how increased complexity in relatively simple chemical systems leads to organization of the material and consequently to chemical evolution. A possible link between prebiotic chemistry and the postulated RNA world is discussed. Received: 12 September 1997 / Accepted: 24 November 1997     

A phylogenetic analysis of cattle DRB3 alleles with a deletion of codon 65

 One of the most common cattle major histocompatibility complex DRB3 alleles, ^* 0201, includes a deletion of codon 65 encoding one residue in the α-helical chain. The mutation is functionally interesting and is likely to influence peptide binding. Exon 2 of two additional del65 alleles, ^* 3301 and ^* 4101, have now been sequenced with the aim to investigate the evolutionary relationship of this allelic group. Despite a fairly large genetic distance between the three alleles (11–17 nucleotide substitutions causing 8–11 amino acid substitutions) we found clear indications of a common ancestry. The α-helical region was very similar or identical among the alleles whereas the β-strand region was quite divergent. The results indicated that interallelic recombination has contributed to the diversification of the del65 group. Deletion of codon 65 has also been found in a roe deer DRB1 allele and a cattle DQB3 allele. Sequence comparisons of the cattle and roe deer DRB del65 alleles refuted the possibility of a trans-species persistence of a del65 allelic lineage but the two species may share a short ancestral sequence motif including del65. In addition to del65, the cattle DQB3 allele did not show any striking sequence similarities to the DRB alleles. Received: 20 March 1997 / Revised: 17 June 1997