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PubMedDate not supplied

Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects.

Gene product distribution is often used to infer developmental similarities and differences in animals with evolutionarily diverse body plans. However, to address commonalties of developmental mechanisms, what is really needed is a method to assess and compare gene function in divergent organisms. This requires mutations eliminating gene function. Such mutations are often difficult to obtain, even in organisms amenable to genetic analysis. To address this issue we have investigated the use of double-stranded RNA interference to phenocopy null mutations. We show that RNA interference can be used to phenocopy mutations of the Deformed orthologues in Drosophila and Tribolium. We discuss the possible use of this technique for comparisons of developmental mechanisms in organisms with differing ontogenies.

Animals↗
PubMed1998

Genetic and biochemical evidence for distinct key functions of two highly divergent GAPDH genes in catabolic and anabolic carbon flow of the cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacterial genomes harbour two separate highly divergent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, gap1 and gap2, which are closely related at the sequence level to the nuclear genes encoding cytosolic and chloroplast GAPDH of higher plants, respectively. Genes gap1 and gap2 of the unicellular cyanobacterium Synechocystis sp. PCC 6803 were cloned and sequenced and subsequently inactivated by insertional mutagenesis to understand their metabolic functions. We obtained homozygous gap1- mutants which have lost the capacity to grow on glucose under dim light while growth on organic acids as well as photosynthetic growth under CO2 and high light is not impaired. Homozygous gap2- mutants show the reciprocal phenotype. Under dim light they only grow on glucose but not on organic acids nor do they survive under photosynthetic conditions. Measurements of the anabolic activities (reduction of 1,3-bisphosphoglycerate) in extracts from wild type and mutant cells show that Gap2 is a major enzyme with dual cosubstrate specificity for NAD and NADP, while Gap1 displays a minor NAD-specific GAPDH activity. However, if measured in the catabolic direction (oxidation of glyceraldehyde-3-phosphate) Gap2 activity is very low and increases three- to fivefold after gel filtration of extracts over Sephadex G25. Our results suggest that enzymes Gap1 and Gap2, although coexpressed in cyanobacterial wild-type cells, play distinct key roles in catabolic and anabolic carbon flow, respectively. While Gap2 operates in the photosynthetic Calvin cycle and in non-photosynthetic gluconeogenesis, Gap1 seems to be essential only for glycolytic glucose breakdown, conditions under which the catabolic activity of Gap2 seems to be repressed by a specific low-molecular-weight inhibitor.

Amino Acid Sequence↗
PubMed2006

Cooperation of sonic hedgehog enhancers in midline expression.

In zebrafish, as in other vertebrates, the secreted signalling molecule Sonic hedgehog (Shh) is expressed in organiser regions such as the embryonic midline and the zona limitans intrathalamica (zli). To investigate the regulatory mechanisms underlying the pattern of shh expression, we carried out a systematic analysis of the intronic regulatory sequences of zebrafish shh using stable transgenesis. Deletion analysis identified the modules responsible for expression in the embryonic shield, the hypothalamus and the zli and confirmed the activities of previously identified notochord and floor plate enhancers. We detected a strong synergism between regulatory regions. The degree of synergy varied over time in the hypothalamus suggesting different mechanisms for initiation and maintenance of expression. Our data show that the pattern of shh expression in the embryonic central nervous system involves an intricate crosstalk of at least 4 different regulatory regions. When compared to the enhancer activities of the mouse Shh gene, we observed a remarkable divergence of function of structurally conserved enhancer sequences. The activating region ar-C (61% identical to SFPE2 in mouse Shh), for example, mediates floor plate expression in the mouse embryo while it directs expression in the forebrain and the notochord and only weakly in the floor plate in the zebrafish embryo. This raises doubts on the predictive power of phylogenetic footprinting and indicates a stunning divergence of function of structurally conserved regulatory modules during vertebrate evolution.

Animals↗
PubMed1998

In vivo treatment with granulocyte colony-stimulating factor results in divergent effects on neutrophil functions measured in vitro.

We have studied the effects of granulocyte colony-stimulating factor (G-CSF) administration to normal individuals on a variety of functional and biochemical neutrophil characteristics that relate to host defense. G-CSF adversely affected neutrophil (polymorphonuclear leukocyte [PMN]) chemotaxis. While this could be partially explained by reduced assembly of neutrophil F-actin, we also recognized an elevated cytosolic calcium mobilization and a normal upregulation of neutrophil CD11b. G-CSF resulted in reduced PMN killing of Staphylococcus aureus with a 10:1 (bacteria:neutrophil) ratio and normal killing with a 1:1 ratio. In association with this, we demonstrated divergent effects on the respiratory burst of intact cells and divergent effects on the content of marker proteins for neutrophil granules. While G-CSF may have resulted in increased content of cytochrome b558 in the cell membrane, it did not alter the amounts of cytosolic oxidase components. After therapy, there was normal content of the azurophilic granule marker, myeloperoxidase, decreased content of the specific granule marker, lactoferrin, and normal content of lysozyme (found in both granules classes). Finally, G-CSF therapy markedly reduced the apoptotic rate of the isolated neutrophil. Therefore, considering disparate functional and biochemical activities, the real benefit of G-CSF therapy may lie in enhanced number and survival of neutrophils.

Adult↗
PubMed2002

Functional analysis of a divergent system II protein, Ccs1, involved in c-type cytochrome biogenesis.

The Ccs1 gene, encoding a highly divergent novel component of a system II type c-type cytochrome biogenesis pathway, is encoded by the previously defined CCS1 locus in Chlamydomonas reinhardtii. phoA and lacZalpha bacterial topological reporters were used to deduce a topological model of the Synechocystis sp. 6803 Ccs1 homologue, CcsB. CcsB, and therefore by analogy Ccs1, possesses a large soluble lumenal domain at its C terminus that is tethered in the thylakoid membrane by three closely spaced transmembrane domains in the N-terminal portion of the protein. Molecular analysis of ccs1 alleles reveals that the entire C-terminal soluble domain is essential for Ccs1 function and that a stromal loop appears to be important in vivo, at least for maintenance of Ccs1. Site-directed mutational analysis reveals that a single histidine (His(274)) within the last transmembrane domain, preceding the large lumenal domain, is required for c-type cytochrome assembly, whereas an invariant cysteine residue (Cys(199)) is shown to be non-essential. Ccs1 is proposed to interact with other Ccs components based on its reduced accumulation in ccs2, ccs3, ccs4, and ccsA strains.

Alleles↗
PubMed1999

Action (verb naming) fluency as an executive function measure: convergent and divergent evidence of validity.

Numerous studies have demonstrated dissociable neuroanatomic underpinnings for the retrieval of grammatical classes of words such as nouns and verbs. Whereas retrieval of common and proper nouns is primarily mediated by posterior and anterior temporal regions, respectively, verb retrieval is primarily mediated by frontal regions. The majority of studies evaluating verb production have utilized tasks requiring subjects to name a graphically depicted action (i.e. action naming), leaving tests of verb generation in the absence of prompting stimuli (i.e. action fluency) largely unexamined. In a recent study, Piatt, Fields, Paolo, Koller and Tröster (in press) found that an action fluency task discriminated demented Parkinson's disease (PD) patients from non-demented PD patients and healthy control subjects, whereas lexical and categorical fluency tasks did not. These authors suggested that action fluency was sensitive to the fronto-striatal pathophysiology associated with PD dementia, and thus, that action fluency might serve as an indicator of executive functioning. This study was undertaken to evaluate the construct validity of action fluency as an executive function measure in a group of healthy elderly control subjects. Findings revealed modest to moderate relationships between action fluency and several putative executive measures. Action fluency was unrelated to indices of semantic and episodic memory. Results support the construct validity of action fluency as an executive function measure and suggest that this task may provide some unique information not tapped by traditional executive function tasks.

Aged↗
PubMed2000

Robust mRNA transcription in chicken DT40 cells depleted of TAF(II)31 suggests both functional degeneracy and evolutionary divergence.

We have employed gene targeting coupled with conditional expression to construct a chicken DT40 cell line in which a tetracycline (Tet)-repressible promoter is exclusively responsible for expression of cTAF(II)31, a histone-like TAF(II) residing in both the transcription factor TFIID and the histone acetylase complex PCAF/SAGA. Tet addition resulted in rapid loss of cTAF(II)31 mRNA and protein, eventually leading to apoptotic cell death. Significantly, five of six other TAF(II)s tested were also rapidly depleted, but levels of the TATA binding protein and subunits of PCAF/SAGA were at most modestly compromised. Strikingly, pulse-labeling experiments indicate that total poly(A)(+) mRNA transcription was not significantly reduced after cTAF(II)31 depletion, and steady-state levels of several specific transcripts remained the same or decreased only mildly. Moreover, activation of c-fos transcription following serum starvation occurred efficiently in the absence of cTAF(II)31. These data, which contrast with comparable studies in yeast, strongly suggest that cTAF(II)31 and perhaps other TAF(II)s are not essential for general mRNA transcription in DT40 cells. We propose that this is due to extensive functional degeneracy in the highly complex metazoan transcriptional machinery.

Acetyltransferases↗
PubMed1996

Conservation of the genetic switch between replication and transfer genes of IncP plasmids but divergence of the replication functions which are major host-range determinants.

The trfA operon of broad-host-range IncP plasmids is essential to activate the origin of vegetative replication in diverse species. The trb operon encodes most of the apparatus for mating pair formation, the first step in conjugative transfer. Comparison of the nucleotide sequence of the IncP beta plasmid R751 presented here with the equivalent IncP alpha sequence identifies conserved features of the organization and regulation of the trfA operon and the region controlling expression of the trb operon. As in IncP alpha plasmids, these operons are transcribed from a bidirectional promoter region consisting of trfAp for the trfA operon and trbAp and trbBp for the trb operon. The KorA-dependent switch between the trfA and trbA promoters is conserved as is the trbA gene encoding the third IncP global regulator. The intergenic region between trbA and trbB shows very little sequence identity between the two plasmids but the spacing, the KorB operator, the trbB promoter, and the existence of a hairpin loop (albeit of different actual sequence) which sequesters the trbB ribosome binding site are all conserved. The trfA operon encodes two ORFs. The first ORF is highly conserved and encodes a putative single-stranded DNA binding protein (Ssb). The second, trfA, contains two translational starts as in the IncP alpha plasmids, generating related polypeptides of 406 (TrfA1) and 282 (TrfA2) amino acids. TrfA2 is very similar to the IncP alpha product, whereas the N-terminal region of TrfA1 shows very little similarity to the equivalent region of IncP alpha TrfA1. This region has been implicated in the ability of IncP alpha plasmids to replicate efficiently in Pseudomonas aeruginosa. A TcR derivative of R751 was constructed and shown not to establish itself efficiently in P. aeruginosa at 37 degrees C, although it did establish itself inefficiently at lower temperatures, underlining the importance of this region in the adaptation of the plasmid to the host.

Amino Acid Sequence↗
PubMed2026

Functional convergence of regulatory regions provides vital insights into mammalian gliding adaptation.

Uncovering the key genetic basis of complex phenotypic convergence in distantly related species has been a long-standing focus in evolutionary biology and genetics, and the convergent evolution of gliding in mammals offers a valuable opportunity to address this question. Here, we investigated the genomic basis of convergent evolution of gliding in mammals by analyzing both protein-coding genes and conserved non-coding elements (CNEs). We first de novo assembled and annotated two chromosome-level genomes of gliding mammals, the red and white giant flying squirrel (Petaurista alborufus) and sugar gliders (Petaurus breviceps), and conducted comprehensive comparative genomic analysis combined with another gliding mammal, the Sunda flying lemur (Galeopterus variegatus) and 14 background species. We found that the convergent evolution of protein-coding genes provided relatively limited but functionally relevant evidence linked to gliding phenotypes. By contrast, we found that gliding-accelerated CNEs (GACNEs) cluster near functionally equivalent genes and frequently aggregate into highly diverged yet functionally convergent hotspot regions. Across the three gliding lineages, both GACNEs and hotspot GACNEs show strong convergence in their functional enrichment profiles, suggesting a broad genetic basis underlying the convergent gliding phenotype. Furthermore, we identified 72 core transcription factors underpinning the genetic basis of gliding convergence, including EMX2 and ZFHX3, potentially involved in multiple aspects of gliding adaptation. Our study highlights the role of functional convergence in regulatory regions as a key mechanism in mammalian gliding convergence, offering valuable insights and strategies for uncovering the genetic basis of complex convergent traits, thereby advancing understanding of the molecular basis of convergent traits.

Petaurista alborufus↗
PubMed1991

Convergent evolution of similar function in two structurally divergent enzymes.

An example of two related enzymes that catalyse similar reactions but possess different active sites is provided by comparing the structure of Escherichia coli thioredoxin reductase with glutathione reductase. Both are dimeric enzymes that catalyse the reduction of disulphides by pyridine nucleotides through an enzyme disulphide and a flavin. Human glutathione reductase contains four structural domains within each molecule: the flavin-adenine dinucleotide (FAD)- and nicotinamide-adenine dinucleotide phosphate (NADPH)-binding domains, the 'central' domain and the C-terminal domain that provides the dimer interface and part of the active site. Although both enzymes share the same catalytic mechanism and similar tertiary structures, their active sites do not resemble each other. We have determined the crystal structure of E. coli thioredoxin reductase at 2 A resolution, and show that thioredoxin reductase lacks the domain that provides the dimer interface in glutathione reductase, and forms a completely different dimeric structure. The catalytically active disulphides are located in different domains on opposite sides of the flavin ring system. This suggests that these enzymes diverged from an ancestral nucleotide-binding protein and acquired their disulphide reductase activities independently.

Amino Acid Sequence↗
PubMed1997

Moderate and severe nutrient restriction has divergent effects on gonadotroph function in orchidectomized sheep.

The effect of plane of nutrition and estradiol (E2) on pituitary concentrations of GnRH receptor and GnRH receptor mRNA was assessed in orchidectomized sheep (wethers). As detailed in the companion paper, 36 wethers were fed to gain, maintain, or lose body weight. Six animals from each feeding group received E2 (0.31 microg E2/50 kg per h) or vehicle during Days 51-54 of controlled feeding. Anterior pituitary tissue was collected at the end of infusion. Both moderate and severe nutrient restriction increased (p < 0.05) tissue concentrations of FSH and FSHbeta mRNA. Conversely, concentrations of GnRH receptor and receptor mRNA were not affected (p > 0.05) by plane of nutrition. Estradiol increased (p < 0.05) GnRH receptor and receptor mRNA in wethers fed to gain or maintain weight. However, this E2-induced response was not evident in wethers subject to severe nutrient restriction. These data demonstrate that severe, but not moderate, nutrient restriction suppresses E2-induced augmentation of tissue concentrations of GnRH receptor and GnRH receptor mRNA. Collectively, the data presented here and in the companion paper suggest that severe nutrient restriction leads to physiologic changes that render the hypothalamus increasingly sensitive to estrogenic stimulation, while the pituitary is made less responsive to steroidal inputs.

Animal Nutritional Physiological Phenomena↗
PubMed1993

Divergence between cytotoxic effector function and tumor necrosis factor alpha production for inflammatory CD4+ T cells from mice with Sendai virus pneumonia.

Sendai virus pneumonia in beta 2-microglobulin-deficient [beta 2-m(-/-)] mice lacking CD8+ T cells is characterized by the development of CD4+ cytotoxic T lymphocytes that can be recovered directly from the respiratory tract. These CD4+ cytotoxic T lymphocytes are not found in beta 2-m (+/+) mice, though inflammatory CD4+ T cells from both beta 2-m (-/-) and beta 2-m (+/+) mice produce substantial amounts of tumor necrosis factor alpha. Blocking experiments with a monoclonal antibody that also inhibits tumor necrosis factor beta show that the secreted forms of these two cytokines are not responsible for virus-specific killing of class II major histocompatibility complex-compatible targets. Comparison of electron micrographs indicates that the CD4+ effectors from the beta 2-m (-/-) mice are potent inducers of apoptosis, while this is not the case for the beta 2-m (+/+) CD4+ set. These experiments further define the functional status of virus-specific CD4+ T cells responding in vivo in the presence or absence of CD8+ effectors.

Animals↗
PubMed1990

Divergency of structure and function of vertebrate xanthine:NAD+ oxidoreductase.

Selection pressure for increasing metabolic flux through a define metabolic pathway affects the enzyme levels, enzyme structure and their kinetic properties. These aspects exemplified by xanthine oxidoreductase from vertebrates of various type of nitrogen excretion are discussed. Two trends in evolutionary kinetic changes of oxypurine hydroxylating activity could be distinguished. Changes in the subunit structure and kinetic properties suggest that the domain catalysing oxypurine hydroxylation and the one cooperating with NAD+ evolved through separate pathways.

Animals↗