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[Coagulation, inflammation and immune response -- an evolutionary conserved plan as cause for disseminated intravasal coagulation?].

The concomitant activation of the immune response and the coagulation system in response to injury represents a phylogenetic old and adaptive principle already present in the early eukaryotic development. The close connection between coagulation, inflammation, and immune defense has been conserved during evolution and can still be found in humans in a number of physiologic reactions to potentially detrimental effects.

Biological Evolution↗

Characterization of the transcription factor MTF-1 from the Japanese pufferfish (Fugu rubripes) reveals evolutionary conservation of heavy metal stress response.

The pufferfish Fugu rubripes was recently introduced as a new model organism for genomic studies, since it contains a full set of vertebrate genes but only 13% as much DNA as a mammal. Fugu genes tend to be smaller and densely spaced due to shortening of introns and intergenic spacers. We isolated the Fugu gene for the metal-responsive transcription factor MTF-1 (MTF1), a mediator of heavy metal regulation and oxidative stress response previously characterized in mammals. In addition, most of the cDNA sequence was also determined. The 780 amino acid MTF-1 protein of Fugu is very similar to that of mouse and human, with 90% amino acid identity in the DNA binding zinc finger domain and 57% overall identity. Expression of the pufferfish cDNA in mammalian cells shows that Fugu MTF-1 has the same DNA binding specificity as its mammalian counterpart and also induces transcription in response to zinc and cadmium. The protein-coding part of the Fugu MTF-1 gene spans 6.4 kb and consists of 11 exons. Upstream region and first exon constitute a CpG island. The distance between stop codon and polyadenylation motifs is >2 kb, suggesting a very long 3' untranslated mRNA region, followed by another CpG island which may represent the promoter of the next gene downstream. Part of the MTF-1 genomic structure was also determined in the mouse, and some striking similarities were found: for example, the upstream adjacent gene in both species is INPP5P, encoding a phosphatase. The mouse MTF-1 promoter is also embedded in a CpG island, which however shares no sequence similarity to the one of Fugu. The Fugu CpG island is shorter than the one of the mouse and has no elevated [G+C] content; these and other data indicate that CpG islands of fish may represent a primordial stage of CpG island evolution.

Amino Acid Sequence↗

Evolutionary conserved cathepsin E substrate specificity as defined by N-terminal and C-terminal sequencing of peptide pools.

The substrate specificity of the non-lysosomal aspartic protease cathepsin E from three different species has been studied using the method of automated N-terminal sequencing and a newly developed method for C-terminal sequencing of peptides and peptide pools. The combination of N-terminal and C-terminal sequencing of peptide pools is a fast and easy method to identify and compare the substrate specificity of endopeptidases. Our analysis shows a conserved hydrolytic specificity between human, mouse and bovine cathepsin E, with only small differences in fine specificity. Furthermore, our results confirm and extend the rules governing the interactions of the substrate with the amino acid (aa) side chains of the various pockets within the enzyme's active cleft. We found that the positions flanking the scissile peptide bond P1-P1' are occupied exclusively by hydrophobic aa with both aliphatic or aromatic side chains; Val and Ile, however, are not allowed in the S1 binding site. The S2 and S2' subsites accept hydrophilic aa. Additional requirements concerning the S3' to S5' subsites were also revealed. Finally, the sequences of single peptides generated by cathepsin E from the three different species can be easily aligned to the determined cleavage motif, showing the reliability of our pool sequencing methods.

Amino Acid Sequence↗

Sodium and potassium ATPase of the teleost fish Catostomus commersoni. Sequence, protein structure and evolutionary conservation of the alpha-subunit.

The alpha-subunit of a Na+/K+ ATPase has been cloned by analysing a lambda gt11 library constructed from polyA+ RNA from the hypothalamic region of the teleost fish Catostomus commersoni (white sucker). The cDNA clone consists of 3853 bp and predicts a protein of 1027 amino-acid residues. Alignment of the sucker sequence with protein sequences previously published for alpha-subunits from various species reveals a high degree of homology throughout the entire sequence containing five potential sites for N-glycosylation, a phosphorylation site and a site for binding fluorescein 5'-isothiocyanate (FITC). A hydropathy profile predicts a secondary structure of the Na+/K+ ATPase alpha-subunit with at least eight membrane-spanning domains. Northern and southern blot analyses suggest the existence of two distinct Na+/K+ ATPase alpha-subunit genes in the sucker genome.

Amino Acid Sequence↗

Evolutionary conservation of the FLOWERING LOCUS C-mediated vernalization response: evidence from the sugar beet (Beta vulgaris).

In many plant species, exposure to a prolonged period of cold during the winter promotes flowering in the spring, a process termed vernalization. In Arabidopsis thaliana, the vernalization requirement of winter-annual ecotypes is caused by the MADS-box gene FLOWERING LOCUS C (FLC), which is a repressor of flowering. During the vernalization process, FLC is downregulated by alteration of its chromatin structure, thereby permitting flowering to occur. In wheat, a vernalization requirement is imposed by a different repressor of flowering, suggesting that some components of the regulatory network controlling the vernalization response differ between monocots and dicots. The extent to which the molecular mechanisms underlying vernalization have been conserved during the diversification of the angiosperms is not well understood. Using phylogenetic analysis, we identified homologs of FLC in species representing the three major eudicot lineages. FLC homologs have not previously been documented outside the plant family Brassicaceae. We show that the sugar beet FLC homolog BvFL1 functions as a repressor of flowering in transgenic Arabidopsis and is downregulated in response to cold in sugar beet. Cold-induced downregulation of an FLC-like floral repressor may be a central feature of the vernalization response in at least half of eudicot species.

Arabidopsis↗

Evolutionary conservation of the insulin gene structure in invertebrates: cloning of the gene encoding molluscan insulin-related peptide III from Lymnaea stagnalis.

Although insulins and structurally related peptides are found in vertebrates as well as in invertebrates, it is not clear whether the genes encoding these hormones have emerged from a single ancestral (insulin)-type of gene or, alternatively, have arisen independently through convergent evolution from different types of gene. To investigate this issue, we cloned the gene encoding the molluscan insulin-related peptide III (MIP III) from the freshwater snail, Lymnaea stagnalis. The predicted MIP III preprohormone had the overall organization of preproinsulin, with a signal peptide and A and B chains, connected by two putative C peptides. Although MIP III was found to share key features with vertebrate insulins, it also had unique structural characteristics in common with the previously identified MIPs I and II, thus forming a distinct class of MIP peptides within the insulin superfamily. MIP III is synthesized in neurones in the brain. It is encoded by a gene with the overall organization of the vertebrate insulin genes, with three exons and two introns, of which the second intron interrupts the coding region of the C peptides. Our data therefore demonstrate that in the Archaemetazoa, the common ancestor of the vertebrates and invertebrates, a primordial peptide with a two-chain insulin configuration encoded by a primordial insulin-type gene must have been present.

Amino Acid Sequence↗

Homeobox genes exhibit evolutionary conserved regionalization in the central nervous system of an ascidian larva.

Animals in each subgroup of the phylum Chordata exhibit a similar process by which they form a tubular central nervous system (CNS). However, little is known about spatial relationship among the CNSs of chordates; vertebrates, cephalochordates and urochordates (tunicates). Ascidians constitute a major animal group in the subphylum Urochordata. In the present study, we examined the expression patterns of labial and orthodenticle related genes of the ascidian, Halocynthia roretzi, in the developing larval CNS. These homeobox genes exhibited region-specific expression patterns that are strikingly similar to those of murine Hoxb-1 and Otx2. The regionalization as characterized by the expression of these genes supports the division of the ascidian larval CNS suggested by the previous morphological studies. Furthermore, conservation of the expression pattern of the homeobox genes suggests that such regionalization occurred in the CNS of a putative common ancestor of chordates.

Amino Acid Sequence↗

Characterization of the mouse PDGF A-chain gene. Evolutionary conservation of gene structure, nucleotide sequence and alternative splicing.

The mouse platelet-derived growth factor (PDGF) A-chain gene has been structurally characterized and compared with its human counterpart. The organization of the two genes is similar. Both consist of 7 exons spaced by 6 introns of corresponding sizes. As in the human gene, exon 6 encodes a sequence which is alternatively spliced. When present, it codes for an alternative C-terminus of the A-chain. In intron 5, conserved stretches of nucleotides, potentially involved in the regulation of the alternative splicing, are identified. The untranslated sequences show a high degree of nucleotide sequence identity and several conserved consensus binding sites for transcription factors are identified within the 5' untranslated as well as in the 5' flanking region.

Alternative Splicing↗

A notable example of an evolutionary conserved gene: studies on a putative DNA helicase TIP49.

TIP49a (just called as simply TIP49 in previous reports [Kanemaki et al., 1997; Makino et al., 1998]) was found in a rat nuclear protein complex that included the TATA-binding protein. TIP49a possesses multiple sequence motifs for ATPase and DNA helicase. Since TIP49a structurally resembles prokaryotic DNA helicase RuvB, TIP49a is resumed to be a putative DNA helicase. We demonstrated TIP49a-related gene(s) in variety organisms from human to archaea. Amino acid identities expressed as aligned scores of human, yeast, and A. fulgidus TIP49a gene counterparts to the rat sequence were 99, 67, and 46, respectively. Strikingly, two homologous regions of mammalian TIP49a and bacterial RuvB exhibited an aligned score of 17-38. We demonstrated that the eukaryotic TIP49a counterparts were immunologically conserved. These lines of evidence show that the TIP49a gene is a notable example of a highly conserved gene among organisms. An extensive homology search revealed another class of TIP49-related gene in the eukaryotes, designated as TIP49b. Moreover, a phylogenetical study suggested that archaeal TIP49 genes belong to the TIP49b ancestor but not to the TIP49a one and that TIP49a evolved from TIP49b in accordance with divergence of archaea and eukarya. The TIP49 gene family is thought to play a fundamental role in a biological activity.

ATPases Associated with Diverse Cellular Activitie↗

Evolutionary conservation of a gene which controls the VH regions of an anti-hapten antibody (NP).

Wild mice of eight types were immunized with hapten NP, and the occurrence of a genetically controlled idiotype NP-b in their sera was studied. Five of the mouse types were subspecies of Mus musculus, the others were M. cervicolor, M. dunni and M. caroli. An idiotype indistinguishable from NP-b was the prediminant anti-NP antibody in M. m. molossinus, the other seven types of mice did not have this antibody population. Lack of the idiotype in these mice does not show that they do not have the VH gene controlling its expression since the idiotypic population must have the lambda light chain, and the seven types of mice either have little of lambda immunoglobulin or have lambda chains different from the lambda chains of the laboratory mice.

Animals↗

Comprehensive analysis of all triple helical repeats in beta-spectrins reveals patterns of selective evolutionary conservation.

The spectrin superfamily (spectrin, alpha-actinin, utrophin and dystrophin) has in common a triple helical repeating unit of ~106 amino acid residues. In spectrin, alpha and beta chains contain multiple copies of this repeat. beta-spectrin chains contain the majority of binding activities in spectrin and are essential for animal life. Canonical beta-spectrins have 17 repeats; beta-heavy spectrins have 30. Here, the repeats of five human beta-spectrins, plus beta-spectrins from several other vertebrates and invertebrates, have been analysed. Repeats 1, 2, 14 and 17 in canonical beta are highly conserved between invertebrates and vertebrates, and repeat 8 in some isoforms. This is consistent with conservation of critical functions, since repeats 1, 2 and 17 bind alpha-spectrin. Repeats 1 of beta-spectrins are not always detected by SMART or Pfam tools. A profile hidden Markov model of beta-spectrin repeat 1 detects alpha-actinins, but not utrophin or dystrophin. Novel examples of repeat 1 were detected in the spectraplakins MACF1, BPAG1 and plectin close to the actin-binding domain. Ankyrin binds to the C-terminal portion of repeat 14; the high conservation of this entire repeat may point to additional, undiscovered ligand-binding activities. This analysis indicates that the basic triple helical repeat pattern was adapted early in the evolution of the spectrin superfamily to encompass essential binding activities, which characterise individual repeats in proteins extant today.

Amino Acid Sequence↗

[Extreme evolutionary conservation of CaSox4 gene in alligator Alligator sinensis].

The SOX genes form a gene family related by homology to the high-mobility-group (HMG) box region of the testis-determining gene, SRY. Using degenerate primer PCR, we have cloned and sequenced the HMG-box motif of CaSox4 gene from genomic DNA of the Chinese alligator, Alligator sinensis, a species closely related to American alligator, Alligator mississippiensis. Sequence comparison showed that CaSox4 gene shared 97% homology at the nucleotide level and complete identity at the amino acid level, respectively, with AES4 gene of Alligator mississippiensis. It indicates that the Sox4 gene has been extremely conserved during evolution. The result obtained in the present study strongly suggests that the Sox4 gene did not diverge during the differentiation of two closely related alligators, which are thought to have originated from a common ancestor and have existed largely unchanged for 200-250 Myr.

Alligators and Crocodiles↗

Differential expression of mRNAs for neuropeptide Y-related peptides in rat nervous tissues: possible evolutionary conservation.

Neuropeptide Y (NPY) is the only member of its peptide family that has been isolated from the mammalian CNS. We have recently found that two different NPY-related molecules are present in the CNS of a cyclostome, the river lamprey (Lampetra fluviatilis) (Söderberg et al., 1991). Here we show that this is also true for the rat CNS, by demonstrating expression of peptide YY (PYY) mRNA in brainstem neurons distinct from those neurons that express NPY mRNA. Dissimilar oligonucleotide DNA probes complementary to 3' untranslated regions of the rat PYY, NPY, and pancreatic polypeptide (PP) mRNA were used in in situ hybridization experiments on sections of rat brain and spinal cord, visceral organs, and peripheral nerve ganglia. The PYY probe hybridized with two populations of neurons in the brainstem: one dispersed along the midline in the rostral medulla and another in the lateral caudal medulla (A1 region). No additional labeling was detected in the remainder of the neuraxis. In the periphery, PYY hybridization was seen only in endocrine cells of the colon, and not in sympathetic ganglia or the adrenal gland, suggesting that previous observations of PYY immunoreactivity in these latter structures were due to antibody cross-reactivity with NPY. The NPY probe did not hybridize with cells on the midline region that contains PYY neurons, but it did label large numbers of neurons throughout the neuraxis. No expression of PP mRNA was detected in the CNS. Northern blot analysis failed to detect PYY mRNA in the CNS, further supporting the observation that PYY is only expressed by a discrete collection of CNS neurons. The anatomy of PYY- and NPY-expressing cells in the CNS and gut shows a striking similarity between rat and lamprey (Brodin et al., 1989), vertebrates that diverged evolutionarily about 450 million years ago, suggesting that both peptide systems have been conserved throughout vertebrate evolution.

Animals↗

[The e(y)3 gene codes for SAYP, an evolutionary conserved protein that is essential for ontogeny].

Enhancers of yellow (e(y)) is a group of genetically and functionally related genes. The proteins encoded by these genes are involved in transcription regulation. The e(y)3 gene under study codes for an ubiquitous nuclear protein, termed SAYP, which has homologs in various multicellular organisms. SAYP contains an AT-hook domain, two PHD domains, and a new evolutionarily conserved domain. SAYP mutants die during embryonic development. The weak e(y)ul mutation causes multiple malformations. The potent e(y)EMSl mutation causes embryonic death, implicating SAYP in early development.

Animals↗

An evolutionary conserved mechanism of T cell activation by microbial toxins. Evidence for different affinities of T cell receptor-toxin interaction.

The enterotoxins produced by Staphylococcus aureus are the most potent mitogens known. They belong to a group of distantly related mitogenic toxins that differ in other biologic activities. In this study we have compared the molecular mechanisms by which these mitogens activate human T lymphocytes. We used the staphylococcal enterotoxins A to E, the staphylococcal toxic shock syndrome toxin, the streptococcal erythrogenic toxins A and C (scarlet fever toxins, erythrogenic toxins (ET)A, ETC), and the soluble mitogen produced by Mycoplasma arthritidis. We found that all these toxins can activate both CD4+ and CD8+ T cells and require MHC class II expression on accessory and target cells. However, T cells could be activated in the absence of class II molecules if the toxins ETA or SEB were co-cross-linked on beads together with anti-CD8 or anti-CD2 antibodies. Enterotoxins, toxic shock syndrome toxin and scarlet toxins stimulate a major fraction of human T cells, and show preferential, but not exclusive, stimulation of T cells carrying certain TCR V beta. In contrast, the mitogen of M. arthritidis, a pathogen for rodents stimulates only a minority of human T cells but activates a major fraction of murine T cells. Analysis of human T cell clones expressing V beta 5 or V beta 8 TCR showed that these clones responded also to those toxins that did not stimulate V beta 5+ and V beta 8+ T cells in bulk cultures. These results indicate that different TCR bind to these toxins with different affinities and that the specificity of the TCR-V beta-toxin interaction is quantitative rather than qualitative in nature. Taken together our findings suggest that these toxins use a common mechanism of T cell activation. They are functionally bivalent proteins crosslinking MHC class II molecules with variable parts of the TCR. Besides V beta, other parts of the TCR must be involved in this binding. The finding that murine T cells responded more weakly to the toxins produced by the human-pathogenic bacteria than to the Mycoplasma mitogen could indicate that the toxins have been adapted to the host's immune system in evolution.

Animals↗

Structural organization of the genes for murine and human leukemia inhibitory factor. Evolutionary conservation of coding and non-coding regions.

Leukemia inhibitory factor, LIF, is a glycoprotein with multiple activities in both the adult and the embryo. LIF appears to be encoded by a unique gene in both mouse and man, although the 3'-untranslated region of the mouse LIF gene gives a complex hybridization pattern on Southern blots. The complete nucleotide sequences of both the murine and human LIF genes and their flanking regions (8.7 and 7.6 kilobase pairs, respectively) were determined and compared. Both genes comprise three exons, two introns and an unusually long 3'-untranslated region (3.2 kilobase pairs), specificying a mRNA of approximately 4.1 kilobases. Two start sites of LIF-transcription were determined, by S1-nuclease protection and by a novel approach involving the polymerase chain reaction. S1-nuclease protection revealed a start site 60-64 base pairs upstream of the translational start codon and immediately downstream of a TATA box (TATATAAAT). The PCR approach identified a second transcriptional start site 160 base pairs 5' of the start codon and adjacent to a "TATA-like" element (CATAATTT). A comparison of the murine and human LIF gene sequences revealed a high degree of conservation in the coding regions and in segments of the untranslated and flanking regions. Seven segments displaying greater than 75% homology were identified, with the 5' and 3' ends of the transcription unit revealing the highest degree of homology. These conserved regions represents potential cis-acting control elements.

Amino Acid Sequence↗