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Expression of foreign genes in lamprey embryos: an approach to study evolutionary changes in gene regulation.

Evolution in development can be viewed as a sequence of changes in gene regulation. To investigate the cross-species compatibility of 5' upstream regulatory regions, we introduced exogenous gene constructs derived from a gnathostome genome into fertilized eggs of the Japanese lamprey, Lampetra japonica, a sister group of the gnathostomes. Eggs were injected with gene constructs in which a sequence encoding the green fluorescent protein (GFP) had been located downstream of either a virus promoter or 5' regulatory regions of medaka actin genes. Reporter gene expression was recorded for more than a month starting two days after injection. Although the expression patterns were highly mosaic and differed among individuals, GFP was expressed predominantly in the striated muscles of lamprey embryos when driven by the 5' upstream regions of the medaka muscle actin genes. This implies that a pan-vertebrate muscle-specific gene regulatory mechanism may have evolved before the agnathan/gnathostome divergence. This gene-transfer technique potentially facilitates the visualization of cells in various differentiating tissues throughout development. The introduction of developmental genes of the lamprey or other animals into lamprey embryos is another potentially important application, one that could provide us with information on the evolutionary changes in functions of genes or gene cascades.

Actins↗

Family consent and the pursuit of better medicines through genetic research.

Rapid changes in the science and technology related to genetic research are challenging scientists, health care providers, ethicists, regulators, patient groups, and the pharmaceutical industry to keep pace with ethically grounded, workable guidelines for both the research and clinical applications of human genetics. We describe the genetic research being conducted by one pharmaceutical company (GlaxoSmithKline) and how the company is addressing the ethical, legal, and social issues surrounding this research; discuss an industry working group's attempt to advance pharmacogenetic research by openly addressing and disseminating information on related ethical, legal, and regulatory issues; identify scientific and ethical differences among various types of genetic research; discuss potential implications of family consent on subject privacy and autonomy, data collection, and study conduct; and suggest points to consider when study sponsors, investigators, and ethics committees evaluate research proposals. Public and expert opinion regarding informed consent in genetic research is evolving as a result of increased education, discussion, and understanding of the relevant issues. Five years ago, there was strong support for anonymity in genetic research as a privacy safeguard. Now, an increasingly popular school of thought advocates against anonymity to preserve an individual's ability to withdraw and, if desired, access research results. It is important to recognize this evolution and address consent issues in a reasoned, practical, and consistent way, including input from patients and their families, health care providers, ethicists, scientists, regulatory bodies, research sponsors, and the lay community. Responsibility for assessing issues related to family consent for research should remain with local investigators, ethics boards, and study sponsors. A "one-size-fits-all" perspective in the form of new regulations, for example, would likely be a disservice to all.

Access to Information↗

Drosophila melanogaster poly(A)-binding protein: cDNA cloning reveals an unusually long 3'-untranslated region of the mRNA, also present in other eukaryotic species [corrected].

Two classes of cDNAs encoding the Drosophila melanogaster (poly(A)-binding protein (PABP), which differ in length due to different positions of their respective 3' ends, were isolated by screening an embryonic cDNA library. These cDNAs hybridize to a single chromosomal site at position 55B on the right arm of the second chromosome. A unique 3.8-kb PABP mRNA species was detected, indicating that 'long' cDNAs correspond to full-length cDNAs and that the 3'-untranslated region of the D. melanogaster mRNA is close to 1.5 kb long. The PABP transcript accumulates in oocytes, is maternally inherited by the embryo and present at every other developmental stage tested. The D. melanogaster PABP cDNAs contain a 1722-nt ORF encoding a 64-kDa protein. This protein contains four RNA-binding domains which show limited primary sequence divergence during evolution, in contrast to the C-terminal third of the protein. The strikingly long 3'-untranslated region of the D. melanogaster PABP mRNA is shown to exist also in other eukaryotes including vertebrate species. It suggests that important regulatory sequences intrinsic to the PABP mRNA are present within this 3'-untranslated region.

Amino Acid Sequence↗

Eye evolution: a question of genetic promiscuity.

Animal eyes have long served as a classical example of independent origin followed by convergence of structures onto a few different solutions. During the past decade this view has been challenged by the discovery of shared developmental regulatory genes. The Pax6 gene in particular is almost universally employed for eye formation in bilaterian animals, despite widely different embryological origins. The resulting controversy on the multiple or single origins of animal eyes has gradually been sharpened by continuing discoveries of further general similarities in the genetic regulatory circuits of eye development. Recent work on gene expression in specified cell types, together with comparative studies of developmental genes in cnidarians, now show some promise to a solution of the controversy.

Animals↗

Purification and cDNA cloning of evolutionally conserved larval cuticle proteins of the silkworm, Bombyx mori.

A specific set of structural proteins termed larval cuticle proteins (LCPs) accumulates in integuments during larval development of the silkworm, Bombyx mori. Two major larval cuticle proteins, LCP17 and LCP22, were purified from the guanidine hydrochloride extract of the larval cuticle, and specific antibodies were raised against these proteins. Immunoblot analysis revealed that both LCPs are actively synthesized during larval intermolt stages, whereas the LCP17 epitope is also slightly but significantly detectable in pupal integuments. cDNA clones for LCPs were isolated by immunoscreening of the cDNA expression library constructed from larval epidermal mRNA. Predicted amino acid sequences of LCP17 and LCP22 are homologous to cuticle proteins from other insect species, including Manduca sexta, Drosophila melanogaster and Locusta migratoria. This fact suggests that these cuticle protein genes originated from a common ancestral gene and have been conserved during evolution. Northern blot hybridization demonstrated that the expression of LCP17 as well as LCP22 mRNA is controlled in a stage-specific manner in the epidermis of the final instar larvae, suggesting a common regulatory mechanism for transcription of these two intermolt genes.

Amino Acid Sequence↗

Pi class glutathione S-transferase genes are regulated by Nrf 2 through an evolutionarily conserved regulatory element in zebrafish.

Pi class GSTs (glutathione S-transferases) are a member of the vertebrate GST family of proteins that catalyse the conjugation of GSH to electrophilic compounds. The expression of Pi class GST genes can be induced by exposure to electrophiles. We demonstrated previously that the transcription factor Nrf 2 (NF-E2 p45-related factor 2) mediates this induction, not only in mammals, but also in fish. In the present study, we have isolated the genomic region of zebrafish containing the genes gstp1 and gstp2. The regulatory regions of zebrafish gstp1 and gstp2 have been examined by GFP (green fluorescent protein)-reporter gene analyses using microinjection into zebrafish embryos. Deletion and point-mutation analyses of the gstp1 promoter showed that an ARE (antioxidant-responsive element)-like sequence is located 50 bp upstream of the transcription initiation site which is essential for Nrf 2 transactivation. Using EMSA (electrophoretic mobility-shift assay) analysis we showed that zebrafish Nrf 2-MafK heterodimer specifically bound to this sequence. All the vertebrate Pi class GST genes harbour a similar ARE-like sequence in their promoter regions. We propose that this sequence is a conserved target site for Nrf 2 in the Pi class GST genes.

Amino Acid Sequence↗

Modular multidomain phosphoryl transfer proteins of bacteria.

Recent phylogenetic and structural analyses of multidomain phosphoryl transfer proteins of bacteria have revealed that interdomain (but not intradomain) splicing and fusion, as well as domain duplication and deletion, have occurred frequently during evolution. These events have been found to be exceedingly rare in certain other protein families. Domain-shuffling events are illustrated by examples from the superfamilies of phosphoenolpyruvate-dependent sugar phosphotransferase systems, their transcriptional regulatory protein targets of phosphorylation, sensor autokinase/response regulator signal transduction systems, and permeases of the ATP-binding-cassette type.

Bacterial Proteins↗

The complete nucleotide sequence of the Rattus norvegicus mitochondrial genome: cryptic signals revealed by comparative analysis between vertebrates.

This paper reports the nucleotide sequence of rat mitochondrial DNA, only the fourth mammalian mitochondrial genome to be completely sequenced. Extensive comparative studies performed with similar genomes from other organisms revealed a number of interesting features. 1) Messenger RNA genes: the codon strategy is mainly dictated by the base compositional constraints of the corresponding codogenic DNA strand. The usage of the initiation and termination codons follows well-established rules. In general the canonical initiator, ATG, and terminators, TAA and TAG (in rat, only TAA), are always present when there is gene overlapping or when the mRNAs possess untranslated nucleotides at the 5' or 3' ends. 2) Transfer RNA genes: a number of features suggest the peculiar evolutionary behavior of this class of genes and confirm their role in the duplication and rearrangement processes that took place in the evolution of the animal mitochondrial genome. 3) Ribosomal RNA genes: accurate sequence analysis revealed a number of significant examples of complementarity between ribosomal and messenger RNAs. This suggests that they might play an important role in the regulation of mitochondrial translation and transcription mechanisms. The properties revealed by our work shed new light on the organization and evolution of the vertebrate mitochondrial genome and more importantly open up the way to clearly aimed experimental studies of the regulatory mechanisms in mitochondria.

Amino Acid Sequence↗

The complex genetic locus polyhomeotic in Drosophila melanogaster potentially encodes two homologous zinc-finger proteins.

Differential expression of the homeotic gene complexes, ANT-C and BX-C, of Drosophila melanogaster is partly controlled by trans-regulating factors located outside the two complexes. The complex genetic locus, polyhomeotic (ph), is one of these trans regulators required during development for correct expression of the homeotic selector genes. The ph locus comprises two genetically independent units whose functions are largely redundant. There are two duplicated sequences arranged as a tandem repeat in the ph region, defining two molecular ph units. Sequence analysis of the 28.6 kb of DNA comprising the locus shows varying degrees of sequence conservation between these two molecular units. Long open reading frames with a high degree of conservation have been localized in each tandem repeat. Putative protein products encoded by both the proximal and the distal unit contain several identical or practically identical protein domains: a zinc-finger-forming motif, an alpha-helix motif, a domain rich in serine and threonine residues and stretches of glutamine residues. The presence of these protein domains supports the hypothesis that ph encodes a transcription factor that may function as part of a protein complex. Possible molecular mechanisms leading to the particular structure of the locus are discussed.

Amino Acid Sequence↗

Ciona intestinalis as a model for cardiac development.

The primitive chordate Ciona intestinalis has emerged as a significant model system for the study of heart development. The Ciona embryo employs a conserved heart gene network in the context of extremely low cell numbers and reduced genetic redundancy. Here, I review recent studies on the molecular genetics of Ciona cardiogenesis as well as classic work on heart anatomy and physiology. I also discuss the potential of employing Ciona to decipher a comprehensive chordate gene network and to determine how this network controls heart morphogenesis.

Animals↗

Evolution of the autosomal chorion cluster in Drosophila. III. Comparison of the s18 gene in evolutionarily distant species and heterospecific control of chorion gene amplification.

We present a total of 6.1 x 10(3) base-pairs of DNA sequences, encompassing the s18 gene and flanking regions within the autosomal chorion cluster of three Drosophila species. Against a background of extensive divergence in the intron and even in parts of the coding region, islands of strong sequence conservation are evident. These are particularly notable in the 5' flanking DNA where they extend to approximately -600 base-pairs from the transcription start site. The most conserved segment of the entire chorion cluster is 71 base-pairs in the s18 5' flanking DNA, which in D. melanogaster is part of a region defined functionally as containing amplification control elements (ACE3 region). Transformation analysis, using chimeric transposons of D. melanogaster and D. grimshawi DNA, revealed that amplification control elements of D. grimshawi can support amplification in D. melanogaster. The functionally defined ACE3 region of D. grimshawi includes the conserved 71 base-pair segment, but also non-conserved sequences further upstream, which apparently enhance amplification.

Amino Acid Sequence↗

Two-domain hemoglobin gene of the water flea Moina macrocopa: duplication in the ancestral Cladocera, diversification, and loss of a bridge intron.

Two cDNAs encoding the two-domain hemoglobin (Hb) chains of a crustacean Cladocera, Moina macrocopa, were cloned and their nucleotide (nt) sequences were determined. The amino acid (aa) sequences of both the gene products deduced from the nt sequences consisted of 348 residues and showed 98% identity with each other. These sequences together with the NH(2)-terminal aa sequences of the Hb chains determined after separation by two-dimensional gel electrophoresis showed that the Hb chains are synthesized as a secretory precursor with a signal peptide of 17 aa residues. The aa sequences of M. macrocopa Hb chains shared the following features with those of Daphnia Hb chains. Firstly, the signal peptide is followed by an NH(2)-terminal extension containing a threonine-rich sequence that might play a role in the multimerization of subunit chains. Secondly, the identity between the aa sequences of the first and second domains is exceptionally low. These facts suggest that duplication of the cladoceran Hb gene occurred before the divergence of families Moinidae and Daphniidae. Analysis of genomic DNA showed that the M. macrocopa Hb genes consist of two large repeated regions, encoding the first and second domains of Hb chains, respectively. The intron-exon organization of the first region of the M. macrocopa Hb genes was similar to that found in the Daphnia Hb genes, having the three-exon, two-intron structure characteristic of animal Hb genes. However, the intron bridging the two regions and the most downstream intron in the second region were missing in the Moina genes, providing a new example of intron loss. The following elements in the 5' flanking region were conserved in the Moina and Daphnia genes: (1) TATAAA, a typical TATA box sequence accompanied by a downstream sequence, GAAXAGCATCAGTT (the fourth residue X was G or A in Daphnia and absent in Moina); (2) CCAAT boxes, located upstream of the TATA box; (3) the binding sites for HIF-1 and GATA-1, also located upstream of the TATA box, that may be responsible for up-regulation of the cladoceran Hb genes under hypoxia.

Amino Acid Sequence↗

Mitochondrial DNA reveal that domestic goat (Capra hircus) are genetically affected by two subspecies of bezoar (Capra aegagurus).

This article describes the complete sequences of the mitochondrial DNA displacement loop (D-loop) region and cytochrome b gene from domestic goats in Laos (Laos native) and wild goat "markhor" (C. falconeri). The wild goat "bezoar" (Capra aegagrus) has been considered to be the strongest candidate for the ancestor of the domestic goats (C. hircus); however, there is not sufficient molecular data to verify the hypothesis at present. In phylogenetic analyses, two wild goats, the markhor and the ibex (C. ibex), appeared as an outgroup, while the bezoar was located in a cluster of domestic goats. Mitochondrial haplotypes of Laos natives revealed two distinct major clusters: one was the same as the bezoar, the second, unique to Laos natives. The topology and calibrated levels of sequence divergence suggests that these clusters might represent at least two different subspecies of ancestral bezoars.

Animals↗

Intron recognition comes of AGe.

The molecular basis for the consensus sequence at the 3' ends of introns in higher eukaryotes has now been elucidated. However, this discovery does not explain all aspects of 3' splice site selection.

Animals↗

Sequence analysis and modular organization of threonyl-tRNA synthetase from Thermus thermophilus and its interrelation with threonyl-tRNA synthetases of other origins.

The gene encoding threonyl-tRNA synthetase (Thr-tRNA synthetase) from the extreme thermophilic eubacterium Thermus thermophilus HB8 has been cloned and sequenced. The ORF encodes a polypeptide chain of 659 amino acids (Mr 75 550) that shares strong similarities with other Thr-tRNA synthetases. Comparative analysis with the three-dimensional structure of other subclass IIa synthetases shows it to be organized into four structural modules: two N-terminal modules specific to Thr-tRNA synthetases, a catalytic core and a C-terminal anticodon-binding module. Comparison with the three-dimensional structure of Escherichia coli Thr-tRNA synthetase in complex with tRNAThr enabled identification of the residues involved in substrate binding and catalytic activity. Analysis by atomic absorption spectrometry of the enzyme overexpressed in E. coli revealed the presence in each monomer of one tightly bound zinc atom, which is essential for activity. Despite strong similarites in modular organization, Thr-tRNA synthetases diverge from other subclass IIa synthetases on the basis of their N-terminal extensions. The eubacterial and eukaryotic enzymes possess a large extension folded into two structural domains, N1 and N2, that are not significantly similar to the shorter extension of the archaebacterial enzymes. Investigation of a truncated Thr-tRNA synthetase demonstrated that domain N1 is not essential for tRNA charging. Thr-tRNA synthetase from T. thermophilus is of the eubacterial type, in contrast to other synthetases from this organism, which exhibit archaebacterial characteristics. Alignments show conservation of part of domain N2 in the C-terminal moiety of Ala-tRNA synthetases. Analysis of the nucleotide sequence upstream from the ORF showed the absence of both any anticodon-like stem-loop structure and a loop containing sequences complementary to the anticodon and the CCA end of tRNAThr. This means that the expression of Thr-tRNA synthetase in T. thermophilus is not regulated by the translational and trancriptional mechanisms described for E. coli thrS and Bacillus subtilis thrS and thrZ. Here we discuss our results in the context of evolution of the threonylation systems and of the position of T. thermophilus in the phylogenic tree.

Amino Acid Motifs↗

Role of MR imaging in clinical research studies.

There have been numerous advances in cartilage imaging with magnetic resonance imaging (MRI) over the past several years. However, in the absence of effective treatments for articular cartilage disease, these innovations have had little applicability to clinical practice. Putative new therapies do exist but only in clinical trials aimed at establishing the efficacy and safety of these therapies before they are released into general use. These trials, therefore, represent the earliest opportunity to develop imaging methods specifically for such therapies and the diseases that they treat. Accordingly, it is the commercial, regulatory, and logistical demands of the clinical trials process, rather than those of clinical practice, that ultimately shape the early evolution of these imaging tools. Understanding this process and its priorities is essential to contributing to this development and to keeping radiology in sync with advances in the rest of medicine. The following article reviews this novel pathway for innovation in medical imaging and reflects on how recent advances in cartilage MRI might fit in.

Biomarkers↗

Reconstructing the tempo and mode of evolution in an extinct clade of birds with ancient DNA: the giant moas of New Zealand.

The tempo and mode of evolution of the extinct giant moas of New Zealand remain obscure because the number of lineages and their divergence times cannot be estimated reliably by using fossil bone characters only. We therefore extracted ancient DNA from 125 specimens and genetically typed them for a 658-bp mtDNA control region sequence. The sequences detected 14 monophyletic lineages, 9 of which correspond to currently recognized species. One of the newly detected lineages was a genetically divergent form of Megalapteryx originally described as a separate species, two more were lineages of Pachyornis in southern and northeastern New Zealand, and two were basal lineages of South Island Dinornis. When results from genetic typing and previous molecular sexing were combined, at least 33.6% of the specimens were incorrectly classified. We used longer sequences of the control region and nine other mtDNA genes totaling 2,814 base pairs to derive a strongly supported phylogeny of the 14 moa lineages. Molecular dating estimated the most recent common ancestor of moas existed after the Oligocene drowning of New Zealand. However, a cycle of lineage-splitting occurred approximately 4-10 million years ago, when the landmass was fragmented by tectonic and mountain-building events and general cooling of the climate. These events resulted in the geographic isolation of lineages and ecological specialization. The spectacular radiation of moa lineages involved significant changes in body size, shape, and mass and provides another example of the general influence of large-scale paleoenvironmental changes on vertebrate evolutionary history.

Animals↗