The sequences of heat shock protein 40 (DnaJ) homologs provide evidence for a close evolutionary relationship between the Deinococcus- Thermus group and cyanobacteria

The genes encoding for heat shock protein 40 (Hsp40 or DnaJ) homologs were cloned and sequenced from the archaebacterium Halobacterium cutirubrum and the eubacterium Deinococcus proteolyticus to add to sequences from the gene banks. These genes were identified downstream of the Hsp70 (or DnaK) genes in genomic fragments spanning this region and, as in other prokaryotic species, Hsp70-Hsp40 genes are likely part of the same operon. The Hsp40 homolog from D. proteolyticus was found to be lacking a central 204 base pair region present in H. cutirubrum that encodes for the four cysteine-rich domains of the repeat consensus sequence CxxCxGxG (where x is any amino acid), present in most Hsp40 homologs. The available sequences from various archaebacteria, eubacteria, and eukaryotes show that the same deletion is also present in the homologs from Thermus aquaticus and two cyanobacteria, but in no other species tested. This unique deletion and the clustering of homologs from the Deinococcus–Thermus group and cyanobacterial species in the Hsp40 phylogenetic trees suggest a close evolutionary relationship between these groups as was also shown recently for Hsp70 sequences (R.S. Gupta et al., J Bacteriol 179:345–357, 1997). Sequence comparisons indicate that the Hsp40 homologs are not as conserved as the Hsp70 sequences. Phylogenetic analysis provides no reliable information concerning evolutionary relationship between prokaryotes and eukaryotes and their usefulness in this regard is limited. However, in phylogenetic trees based on Hsp40 sequences, the two archaebacterial homologs showed a polyphyletic branching within Gram-positive bacteria, similar to that seen with Hsp70 sequences. Received: 30 January 1997 / Accepted: 22 March 1997     

The Evolution of the Conserved ATPase Domain (CAD): Reconstructing the History of an Ancient Protein Module

The AAA proteins (ATPases Associated with a variety of cellular Activities) are found in eubacterial, archaebacterial, and eukaryotic species and participate in a large number of cellular processes, including protein degradation, vesicle fusion, cell cycle control, and cellular secretory processes. The AAA proteins are characterized by the presence of a 230 to 250-amino acid ATPase domain referred to as the Conserved ATPase Domain or CAD. Phylogenetic analysis of 133 CAD sequences from 38 species reveal that AAA CADs are organized into discrete groups that are related not only in structure but in cellular function. Evolutionary analyses also indicate that the CAD was present in the last common ancestor of eubacteria, archaebacteria, and eukaryotes. The eubacterial CADs are found in metalloproteases, while CAD-containing proteins in the archaebacterial and eukaryotic lineages appear to have diversified by a series of gene duplication events that lead to the establishment of different functional AAA proteins, including proteasomal regulatory, NSF/Sec, and Pas proteins. The phylogeny of the CADs provides the basis for establishing the patterns of evolutionary change that characterize the AAA proteins. Received: 28 January 1997 / Accepted: 8 May 1997     

Phylogenetic Relationships of the Glycolytic Enzyme, Glyceraldehyde-3-Phosphate Dehydrogenase, from Parabasalid Flagellates

Over 90% of the open reading frame of gap genes for glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was obtained with PCR from five species of Parabasala. With gap1 from Trichomonas vaginalis obtained earlier, the data include two sequences each for three species. All sequences were colinear with T. vaginalis gap1 and shared with it as a synapomorphy a 10- to 11-residue insertion not found in any other gap and an S-loop with characteristic features of eubacterial GAPDH. All residues known to be highly conserved in this enzyme were present. The parabasalid sequences formed a robust monophyletic group in phylogenetic reconstructions with distance-based, maximum-parsimony, and maximum-likelihood methods. The two genes of the amphibian commensal, Trichomitus batrachorum, shared a common ancestor with the rest, which separate into two well-supported lineages. T. vaginalis and Tetratrichomonas gallinarum (both representatives of Trichomonadinae) formed one, while Monocercomonas sp. and Tritrichomonas foetus formed the other. These data agreed with and/or were close to published reconstructions based on other macromolecules. They did not support the ancestral position of Monocercomonas sp. proposed on the basis of morphological characteristics but confirmed an early emergence of Trichomitus batrachorum. The sequence pairs obtained from three species indicated either gene duplications subsequent to the divergence of the corresponding lineages or a strong gene conversion later in these lineages. The parabasalid clade was a robust part of the eubacterial radiation of GAPDH and showed no relationships to the clade that contained all other eukaryotic gap genes. The data clearly reveal that the members of this lineage use in their glycolytic pathway a GAPDH species with properties and an evolutionary history that are unique among all eukaryotes studied so far. Received: 28 April 1997 / Accepted: 14 October 1997     

The Origin of Chlorarachniophyte Plastids, as Inferred from Phylogenetic Comparisons of Amino Acid Sequences of EF-Tu

A molecular phylogenetic analysis of elongation factor Tu (EF-Tu) proteins from plastids was performed in an attempt to identify the origin of chlorarachniophyte plastids, which are considered to have evolved from the endosymbiont of a photosynthetic eukaryote. Partial sequences of the genes for plastid EF-Tu proteins (1,080–1,089 bp) were determined for three algae that contain chlorophyll b, namely, Gymnochlora stellata (Chlorarachniophyceae), Bryopsis maxima (Ulvophyceae), and Pyramimonas disomata (Prasinophyceae). The deduced amino acid sequences were used to construct phylogenetic trees of the plastid and bacterial EF-Tu proteins by the maximum likelihood, the maximum parsimony, and the neighbor joining methods. The trees obtained in the present analysis suggest that all plastids that contain chlorophyll b are monophyletic and that the chlorarachniophyte plastids are closely related to those of the Ulvophyceae. The phylogenetic trees also suggest that euglenophyte plastids are closely related to prasinophycean plastids. The results indicate that the chlorarachniophyte plastids evolved from a green algal endosymbiont that was closely related to the Ulvophyceae and that at least two secondary endosymbiotic events have occurred in the lineage of algae with plastids that contain chlorophyll b. Received: 10 March 1997 / Accepted: 28 July 1997     

Molecular Characterization of the Principal Symbiotic Bacteria of the Weevil Sitophilus oryzae: A Peculiar G + C Content of an Endocytobiotic DNA

The principal intracellular symbiotic bacteria of the cereal weevil Sitophilus oryzae were characterized using the sequence of the 16S rDNA gene (rrs gene) and G + C content analysis. Polymerase chain reaction amplification with universal eubacterial primers of the rrs gene showed a single expected sequence of 1,501 bp. Comparison of this sequence with the available database sequences placed the intracellular bacteria of S. oryzae as members of the Enterobacteriaceae family, closely related to the free-living bacteria, Erwinia herbicola and Escherichia coli, and the endocytobiotic bacteria of the tsetse fly and aphids. Moreover, by high-performance liquid chromatography, we measured the genomic G + C content of the S. oryzae principal endocytobiotes (SOPE) as 54%, while the known genomic G + C content of most intracellular bacteria is about 39.5%. Furthermore, based on the third codon position G + C content and the rrs gene G + C content, we demonstrated that most intracellular bacteria except SOPE are A + T biased irrespective of their phylogenetic position. Finally, using the hsp60 gene sequence, the codon usage of SOPE was compared with that of two phylogenetically closely related bacteria: E. coli, a free-living bacterium, and Buchnera aphidicola, the intracellular symbiotic bacteria of aphids. Taken together, these results show a peculiar and distinctly different DNA composition of SOPE with respect to the other obligate intracellular bacteria, and, combined with biological and biochemical data, they elucidate the evolution of symbiosis in S. oryzae. Received: 8 September 1997 / Accepted: 24 October 1997     

Analysis of the Cluster of Ribosomal Protein Genes in the Plastid Genome of a Unicellular Red Alga Cyanidioschyzon merolae: Translocation of the str Cluster as an Early Event in the Rhodophyte-Chromophyte Lineage of Plastid Evolution

The nucleotide sequence of a cluster of ribosomal protein genes in the plastid genome of a unicellular red alga, Cyanidioschyzon merolae, which has been supposed to be the most primitive alga, was determined. The phylogenetic tree inferred from the amino acid sequence of ribosomal proteins of two rhodophytes, a chromophyte, a glaucophyte, two chlorophytes (land plants), a cyanobacterium, and three eubacteria suggested a close relationship between the cyanobacterium Synechocystis PCC6803 and the plastids of various species in the kingdom Plantae, which is consistent with the hypothesis of the endosymbiotic origin of plastids. In this tree, the two species of rhodophytes were grouped with the chromophyte, and the glaucophyte was grouped with the chlorophytes. Analysis of the organization of the genes encoding the ribosomal proteins suggested that the translocation of the str cluster occurred early in the lineage of rhodophytes and chromophytes after these groups had been separated from chlorophytes and glaucophytes. Received: 2 June 1997 / Accepted: 15 July 1997     

Phylogenetic Analysis of Evolutionary Relationships of the Planctomycete Division of the Domain Bacteria Based on Amino Acid Sequences of Elongation Factor Tu

Sequences from the tuf gene coding for the elongation factor EF-Tu were amplified and sequenced from the genomic DNA of Pirellula marina and Isosphaera pallida, two species of bacteria within the order Planctomycetales. A near-complete (1140-bp) sequence was obtained from Pi. marina and a partial (759-bp) sequence was obtained for I. pallida. Alignment of the deduced Pi. marina EF-Tu amino acid sequence against reference sequences demonstrated the presence of a unique 11-amino acid sequence motif not present in any other division of the domain Bacteria. Pi. marina shared the highest percentage amino acid sequence identity with I. pallida but showed only a low percentage identity with other members of the domain Bacteria. This is consistent with the concept of the planctomycetes as a unique division of the Bacteria. Neither primary sequence comparison of EF-Tu nor phylogenetic analysis supports any close relationship between planctomycetes and the chlamydiae, which has previously been postulated on the basis of 16S rRNA. Phylogenetic analysis of aligned EF-Tu amino acid sequences performed using distance, maximum-parsimony, and maximum-likelihood approaches yielded contradictory results with respect to the position of planctomycetes relative to other bacteria. It is hypothesized that long-branch attraction effects due to unequal evolutionary rates and mutational saturation effects may account for some of the contradictions. Received: 21 August 2000 / Accepted: 8 January 2001     

An Alphoid-Like Satellite DNA Sequence Is Present in the Genome of a Lacertid Lizard

A PstI DNA family was isolated from the genome of a lacertid, Lacerta graeca. The 185-bp monomeric unit (pGPS) was cloned and hybridized to DNAs and chromosomes of several lacertid species. The data showed that pGPS hybridizes to the (1) centromeric or pericentromeric heterochromatin of almost all the chromosomes of L. graeca and (2) genomic DNA of species phylogenetically related and unrelated to L. graeca. The presence of pGPS even in species immunologically apart more than 30 million years suggests that this repeated family might be either very ancient or have been conserved during evolution due to its functional role. The latter hypothesis might be supported by the results of sequence analysis which showed some homology with both several alphoid sequences of primates and the CDEIII centromeric sequence of yeast. Segments of the satellite sequence are similar to the mammalian CENP-B box. These observations suggest that pGPS might have a role in determining the centromeric function in lacertid lizards. Received: 6 February 1997 / Accepted: 14 May 1997     

Heat Shock Protein 70 Family: Multiple Sequence Comparisons, Function, and Evolution

The heat shock protein 70 kDa sequences (HSP70) are of great importance as molecular chaperones in protein folding and transport. They are abundant under conditions of cellular stress. They are highly conserved in all domains of life: Archaea, eubacteria, eukaryotes, and organelles (mitochondria, chloroplasts). A multiple alignment of a large collection of these sequences was obtained employing our symmetric-iterative ITERALIGN program (Brocchieri and Karlin 1998). Assessments of conservation are interpreted in evolutionary terms and with respect to functional implications. Many archaeal sequences (methanogens and halophiles) tend to align best with the Gram-positive sequences. These two groups also miss a signature segment [about 25 amino acids (aa) long] present in all other HSP70 species (Gupta and Golding 1993). We observed a second signature sequence of about 4 aa absent from all eukaryotic homologues, significantly aligned in all prokaryotic sequences. Consensus sequences were developed for eight groups [Archaea, Gram-positive, proteobacterial Gram-negative, singular bacteria, mitochondria, plastids, eukaryotic endoplasmic reticulum (ER) isoforms, eukaryotic cytoplasmic isoforms]. All group consensus comparisons tend to summarize better the alignments than do the individual sequence comparisons. The global individual consensus ``matches' 87% with the consensus of consensuses sequence. A functional analysis of the global consensus identifies a (new) highly significant mixed charge cluster proximal to the carboxyl terminus of the sequence highlighting the hypercharge run EEDKKRRER (one-letter aa code used). The individual Archaea and Gram-positive sequences contain a corresponding significant mixed charge cluster in the location of the charge cluster of the consensus sequence. In contrast, the four Gram-negative proteobacterial sequences of the alignment do not have a charge cluster (even at the 5% significance level). All eukaryotic HSP70 sequences have the analogous charge cluster. Strikingly, several of the eukaryotic isoforms show multiple mixed charged clusters. These clusters were interpreted with supporting data related to HSP70 activity in facilitating chaperone, transport, and secretion function. We observed that the consensus contains only a single tryptophan residue and a single conserved cysteine. This is interpreted with respect to the target rule for disaggregating misfolded proteins. The mitochondrial HSP70 connections to bacterial HSP70 are analyzed, suggesting a polyphyletic split of Trypanosoma and Leishmania protist mitochondrial (Mt) homologues separated from Mt-animal/fungal/plant homologues. Moreover, the HSP70 sequences from the amitochondrial Entamoeba histolytica and Trichomonas vaginalis species were analyzed. The E. histolytica HSP70 is most similar to the higher eukaryotic cytoplasmic sequences, with significantly weaker alignments to ER sequences and much diminished matching to all eubacterial, mitochondrial, and chloroplast sequences. This appears to be at variance with the hypothesis that E. histolytica rather recently lost its mitochondrial organelle. T. vaginalis contains two HSP70 sequences, one Mt-like and the second similar to eukaryotic cytoplasmic sequences suggesting two diverse origins. Received: 29 January 1998 / Accepted: 14 May 1998     

Similarity of Structural Features and Evolution of Satellite DNAs from Palorus subdepressus (Coleoptera) and Related Species

A novel highly abundant satellite DNA comprising 20% of the genome has been characterized in Palorus subdepressus (Insecta, Coleoptera). The 72-bp-long monomer sequence is composed of two copies of T₂A₅T octanucleotide alternating with 22-nucleotide-long elements of an inverted repeat. Phylogenetic analysis revealed clustering of monomer sequence variants into two clades. Two types of variants are prevalently organized in an alternating pattern, thus showing a tendency to generate a new complex repeating unit 144 bp in length. Fluorescent in situ hybridization revealed even distribution of the satellite in the region of pericentric heterochromatin of all 20 chromosomes. P. subdepressus satellite sequence is clearly species specific, lacking similarity even with the satellite from congeneric species P. ratzeburgii. However, on the basis of similarity in predicted tertiary structure induced by intrinsic DNA curvature and in repeat length, P. subdepressus satellite can be classified into the same group with satellites from related tenebrionid species P. ratzeburgii, Tenebrio molitor, and T. obscurus. It can be reasonably inferred that repetitive sequences of different origin evolve under constraints to adopt and conserve particular features. Obtained results suggest that the higher-order structure and repeat length, but not the nucleotide sequence itself, are maintained through evolution of these species. Received: 23 April 1997 / Accepted: 11 July 1997     

Identification of Glyoxalase I Sequences in Brassica oleracea and Sporobolus stapfianus: Evidence for Gene Duplication Events

Glyoxalase I (GlxI) is the first of two enzymes involved in the cellular detoxification of methylglyoxal. A recent search of the National Center for Biotechnology Information (NCBI) databases with the protein sequence of Salmonella typhimurium GlxI identified two new hypothetical proteins with unassigned function. These two sequences, from Brassica oleracea and Sporobolus stapfianus, have significant sequence similarity to known GlxI sequences, suggesting that these two open reading frames encode for GlxI in these plants. Interestingly, analysis of these two new sequences indicates that they code for a protein composed of two fused monomers, a situation previously found solely in the yeast GlxI enzymes. Received: 10 May 1997 / Accepted: 15 October 1997     

Evidence for multiple functional copies of the male sex-determining locus, sry, in African murine rodents

Southern hybridization data suggest that the male sex-determining locus, Sry, is often duplicated in rodents. Here we explore DNA sequence evolution of orthologous and paralogous copies of Sry isolated from six species of African murines. PCR amplification followed by direct sequencing revealed from two to four copies of Sry per species. All copies include a long open reading frame, with a stop codon that coincides closely with the stop codon of the house mouse, Mus musculus, a species known to have a single copy of Sry. A phylogenetic analysis suggests that there are at least seven paralogous copies of Sry in this group of rodents. Putative orthologues are identical; sequence divergence among putative paralogues ranges from 1 to 8% (excluding the CAG repeat), with much lower levels of divergence in the high-mobility group (HMG-box) region than in the C-terminal region. A high proportion of nucleotide substitutions in both regions result in amino-acid replacement. The long open reading frame, conserved HMG-box, and pattern of evolution of the putative paralogues suggest that they are functional. Received: 4 October 1996 / Accepted: 17 January 1997     

Low Divergence in rDNA ITS Sequences Among Five Species of Fucus (Phaeophyceae) Suggests a Very Recent Radiation

Sequences from the two ribosomal DNA internal transcribed spacers (ITS1 and ITS2) were compared among five species of Fucus. Based on the present taxon sampling, parsimony analysis showed that Fucus serratus is the sister-group of the remaining Fucus species when Ascophyllum nodosum was used as an outgroup. The topology of the tree was (Fucus serratus (F. lutarius (F. vesiculosus (F. spiralis+F. ceranoides)))). The extremely low variation observed suggests a very recent radiation of the genus which supports the view widely accepted that the Fucales are among the most evolutionarily advanced of the brown algae. We further note that sequence differences between Fucus and Ascophyllum were 28%: this does not rule out the utility of ITS sequences within the Fucaceae. The very low number of informative positions allows to demonstrate empirically that distance matrix methods group on the basis of symplesiomorphies. Received: 17 February 1997 / Accepted: 17 April 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     

Correlation Between Shine–Dalgarno Sequence Conservation and Codon Usage of Bacterial Genes

In this study, we analyzed the correlation between codon usage bias and Shine–Dalgarno (SD) sequence conservation, using complete genome sequences of nine prokaryotes. For codon usage bias, we adopted the codon adaptation index (CAI), which is based on the codon usage preference of genes encoding ribosomal proteins, elongation factors, heat shock proteins, outer membrane proteins, and RNA polymerase subunit proteins. To compute SD sequence conservation, we used SD motif sequences predicted by Tompa and systematically aligned them with 5′UTR sequences. We found that there exists a clear correlation between the CAI values and SD sequence conservation in the genomes of Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and Methanococcus jannaschii, and no relationship is found in M. genitalium, M. pneumoniae, and Synechocystis. That is, genes with higher CAI values tend to have more conserved SD sequences than do genes with lower CAI values in these organisms. Some organisms, such as M. thermoautotrophicum, do not clearly show the correlation. The biological significance of these results is discussed in the context of the translation initiation process and translation efficiency. Received: 22 June 2000 / Accepted: 18 October 2000