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At least 1,405 records · Page 78Linked to original sources
PubMed2006

Comparative genomics of the HOG-signalling system in fungi.

Signal transduction pathways play crucial roles in cellular adaptation to environmental changes. In this study, we employed comparative genomics to analyse the high osmolarity glycerol pathway in fungi. This system contains several signalling modules that are used throughout eukaryotic evolution, such as a mitogen-activated protein kinase and a phosphorelay module. Here we describe the identification of pathway components in 20 fungal species. Although certain proteins proved difficult to identify due to low sequence conservation, a main limitation was incomplete, low coverage genomic sequences and fragmentary genome annotation. Still, the pathway was readily reconstructed in each species, and its architecture could be compared. The most striking difference concerned the Sho1 branch, which frequently does not appear to activate the Hog1 MAPK module, although its components are conserved in all but one species. In addition, two species lacked apparent orthologues for the Sln1 osmosensing histidine kinase. All information gathered has been compiled in an MS Excel sheet, which also contains interactive visualisation tools. In addition to primary sequence analysis, we employed analysis of protein size conservation. Protein size appears to be conserved largely independently from primary sequence and thus provides an additional tool for functional analysis and orthologue identification.

Cluster Analysis↗
PubMed2000

Molecular cloning of the CRM1 gene from Candida albicans.

In a screen for Candida albicans genes capable of supressing a ste20Delta mutation in Saccharomyces cerevisiae, a homologue of the exportin-encoding gene CRM1 was isolated. The CaCRM1 gene codes for a protein of 1079 amino acids with a predicted molecular weight of 124 029 and isoelectric point of 5.04. Crm1p from C. albicans displays significant amino acid sequence homology with Crm1p from Saccharomyces cerevisiae (65% identity, 74% similarity), Schizosaccharomyces pombe (55% identity, 66% similarity), Caenorhabditis elegans (45% identity, 57% similarity), and Homo sapiens (48% identity, 59% similarity). Interestingly, CaCRM1 encodes a threonine rather than a cysteine at position 533 in the conserved central region, suggesting that CaCrm1p is leptomycin B-insensitive, like S. cerevisiae Crm1p. CaCRM1 on a high copy vector can complement a thermosensitive allele of CRM1 (xpo1-1) in S. cerevisiae, showing that CaCrm1p and S. cerevisiae Crm1p are functionally conserved. Southern blot analysis suggests that CaCRM1 is present at a single locus within the C. albicans genome. The nucleotide sequence of the CaCRM1 gene has been deposited at GenBank under Accession No. AF178855.

Amino Acid Sequence↗
PubMed1998

Something fishy in the rat brain: molecular genetics of the hypothalamo-neurohypophysial system.

The brain peptides vasopressin and oxytocin play crucial roles in the regulation of salt and water balance. The genes encoding these neurohormones are regulated by cell-specific and physiological cues, but the molecular mechanisms remain obscure. New strategies, involving the introduction of rat transgenes into rats, are being used to address these issues, but the complexity of the rat genome has hampered progress. By contrast, the pufferfish, Fugu rubripes, has a "junk-free" genome. The oxytocin homologue from Fugu, isotocin, has been introduced into rats and is expressed in oxytocin neurons, where it is upregulated by physiological perturbations that upregulate the oxytocin gene. The Fugu and rat lineages separated 400 million years ago, yet the mechanisms that regulate the isotocin and oxytocin genes have been conserved. Fugu genome analysis and transgenesis in the physiologically tractable rat host are a powerful combination that will enable the identification of fundamental components of the neural systems that control homeostasis.

Animals↗
PubMed2000

The synthetase domains of cobalamin biosynthesis amidotransferases cobB and cobQ belong to a new family of ATP-dependent amidoligases, related to dethiobiotin synthetase.

Phosphotransacetylases of Escherichia coli and several other bacteria contain an additional 350-aa N-terminal fragment that is not required for phosphotransacetylase activity. Sequence analysis of this fragment revealed that it is closely related to a family of ATP-dependent enzymes that also includes dethiobiotin synthetase and the synthetase domains of two amidotransferases involved in cobalamin biosynthesis, cobyrinic acid a,c-diamide synthase (CobB) and cobyric acid synthase (CobQ). Further database searches showed that this enzyme family is also related to the MinD family of ATPases involved in regulation of cell division in bacteria and archaea. Analysis of sequence conservation in the members of this enzyme family using the structure of dethiobiotin synthetase active site as a guide allowed us to suggest a model for the interaction of CobB and CobQ with their respective substrates. CobB and CobQ were also found to contain unusual Triad family (class I) glutamine amidotransferase domains with conserved Cys and His residues, but lacking the Glu residue of the catalytic triad. These results should help in understanding the enzymology of cobalamin biosynthesis and in resolving the role of phosphotransacetylase in regulation of the carbon flow to and from acetate.

Adenosine Triphosphatases↗
PubMed2001

Detection of malignant effusions: comparison of a telomerase assay and cytologic examination.

Telomerase is inactive in most somatic cells, but has been found to be reactivated in a majority of cancers. Our principal goal was to test whether the presence of telomerase activity concurred with positive cytology, and was thus of potential use in detecting cancer cells in effusions. The telomeric repeat amplification protocol (TRAP) assay and cytological examination were performed in a blinded fashion on 91 unselected effusions, for which laboratory processing was done according to standard procedures. In our series, 30% (27/91) of samples were found to be malignant by cytology. Of these, 19 (70%) were also positive in the TRAP assay. Of the 8 telomerase-negative cytology-positive samples, RNA integrity was generally poor, indicating suboptimal sample conservation for molecular analysis. Negative cytology in the presence of telomerase activity was observed in 17 effusions. Of these, 11 were from patients with advanced cancer, and thus a diagnosis of malignant effusion should be suspected. The TRAP assay for telomerase activity holds promise in the analysis of effusions, but its routine use as an adjunct to cytology awaits further confirmation of its positive predictive value.

Adolescent↗
PubMed1996

Kinesin proteins: a phylum of motors for microtubule-based motility.

The cellular processes of transport, division and, possibly, early development all involve microtubule-based motors. Recent work shows that, unexpectedly, many of these cellular functions are carried out by different types of kinesin and kinesin-related motor proteins. The kinesin proteins are a large and rapidly growing family of microtubule-motor proteins that share a 340-amino-acid motor domain. Phylogenetic analysis of the conserved motor domains groups the kinesin proteins into a number of subfamilies, the members of which exhibit a common molecular organization and related functions. The kinesin proteins that belong to different subfamilies differ in their rates and polarity of movement along microtubules, and probably in the particles/organelles that they transport. The kinesins arose early in eukaryotic evolution and gene duplication has allowed functional specialization to occur, resulting in a surprisingly large number of different classes of these proteins adapted for intracellular transport of vesicles and organelles, and for assembly and force generation in the meiotic and mitotic spindles.

Animals↗
PubMed1981

252Californium plasma desorption mass spectrometry.

The mass spectrometry of involatile molecules presents problems not encountered when the molecule can be volatilized and subsequently ionized in the gas phase. The production of ions from a condensed phase is sensitively dependent on the properties of the matrix. The presence of impurities in the sample can totally quench molecular ion formation even though the sample contains a high percentage of the molecule of interest. One of the methods used for the mass spectrometry of involatile molecules is 252Californium plasma desorption mass spectrometry. The basic principles of this method will be presented with examples of its application that include the identification of involatile marine toxins (including the red tide toxins). We shall also introduce a new variation in mass spectrometry, a study of time-dependent effects, a technique that can be used to follow dynamic changes as a result of chemical reactions occurring in the sample at room temperature. This is possible in 252Californium plasma desorption mass spectrometry because the sample is conserved during the analysis.

Californium↗
PubMed2001

Pitfalls in TRAP assay in routine detection of malignancy in effusions.

Telomerase has been found to be reactivated in a majority of cancers but is inactive in most somatic cells. Our principal goal was to determine the potential use of the telomeric repeat amplification protocol (TRAP) assay as marker for malignancy in cytological effusions. The simple selection criterion was the cytological diagnosis, and routine samples were classified into malignant (58 samples) and nonmalignant (233 samples). Of the malignant samples, 44/58 (76%) were positive by TRAP assay. Of the 14 telomerase-negative cytology-positive samples, RNA integrity was poor in 9, indicating suboptimal sample conservation for molecular analysis. In 3 of the remaining 5 samples with a negative TRAP assay, a high number of malignant cells was observed, and these cells might have been telomerase-negative. Thus, the sensitivity of TRAP assay for the presence of malignant cells was about 76%. In the cytologically nonmalignant effusions, the presence of telomerase activity was observed in 24% (55/233). Of these, 6% were highly suspicious for malignancy, 9% were doubtful, and 9% were cytologically nonmalignant effusions confirmed by a follow-up of 12 mo or more. According to these data, the specificity of the TRAP assay to detect tumor cells in effusions ranged only between 82-91%. Our results indicate that, although the TRAP assay is positive in 6-15% of putative malignant effusions, the relatively high number of TRAP false-negative and false-positive cases renders this test unsuitable for routine diagnostic purposes.

Adolescent↗
PubMed1996

Physiological activation of the IgH 3' enhancer in B lineage cells is not blocked by Pax-5.

The mouse 3' enhancer contains a high-affinity binding site for the paired box protein Pax-5. Here, we demonstrate by genomic footprinting that the rat 3' enhancer contains a low-affinity binding site for Pax-5, which is occupied in activated splenic B cells. Thus, binding of Pax-5 to the IgH 3' enhancer appears to be evolutionarily conserved in rodents. Analysis of Pax-5 expression in primary B cells demonstrates that Pax-5 remains expressed after 4 days of lipopolysaccharide (LPS) induction, but is down-regulated in 5-day stimulated cells. Similarly, the expression of Pax-5 is down-regulated in vivo in activated large splenocytes, in contrast to small resting cells. Multimerization of the high-affinity Pax-5 binding site linked to a heterologous reporter gene demonstrates that Pax-5 can function as a transcriptional activator. In contrast, Pax-5 overexpressed in cell lines represses both the mouse and the rat 3' enhancer. Surprinsingly, cross-linking of the IgM receptor in BAL-17 cells containing a stably integrated 3' enhancer-dependent beta globin reporter gene demonstrates that induction of 3' enhancer activity is not blocked by Pax-5. Moreover, stimulation of 3' enhancer beta globin-transgenic splenocytes demonstrate that Pax-5 cannot repress-activation of the 3' enhancer upon LPS induction or CD40 receptor stimulation. Hence, activation of the IgH 3' enhancer occurs independently of changes in Pax-5 gene expression. This indicates that previous studies conducted in vitro may be an oversimplification of the function of Pax-5 and 3' enhancer activity.

Animals↗
PubMed1999

Cloning of two chemokine receptor homologs (CXC-R4 and CC-R7) in rainbow trout Oncorhynchus mykiss.

Two rainbow trout chemokine receptors have been sequenced, with homology to CXC-R4 and CC-R7 molecules. The CXC-R4 sequence consisted of 1681 nucleotides, which translated into a mature protein of 357 amino acids, with 80.7% similarity to human CXC-R4. The CC-R7 sequence consisted of 2287 nucleotides, which translated into a 368-amino acid mature protein with 64.5% similarity to human CC-R7. Both sequences contained seven hydrophobic regions, representing the seven transmembrane domains (TM) typical of G-protein-coupled receptors. Extracellular cysteines, transmembrane prolines, and the DRY motif immediately following TM3 were conserved. Phylogenetic tree analysis revealed a tight clustering of trout CXC-R4 with CXC-R3-5 genes. Trout CC-R7 clustered with CC-R6-7 and CXC-R1-2. Reverse transcriptase-polymerase chain reaction analysis demonstrated a wide tissue distribution of CXC-R4 and CC-R7 message in trout, being present in head-kidney leukocytes, blood, gill, brain, spleen, and liver.

Amino Acid Sequence↗
PubMed1993

Phylogeny of the insulin-like growth factors (IGFs) and receptors: a molecular approach.

The IGFs (IGF-I and IGF-II) are essential for normal mammalian growth and development. Their actions are mediated primarily by their interactions with the type I IGF receptor (IGF-I receptor), a transmembrane tyrosine kinase. The ligands and the IGF-I receptor are structurally related to insulin and to the insulin receptor, respectively. Analysis of evolutionary conservation has often provided insights into essential regions of molecules such as hormones and their receptors. The genes for insulin and IGFs have been partially characterized in a number of vertebrate species extending evolutionarily from humans as far back as fish. The sequences of the exons encoding the mature insulin and IGF peptides are highly conserved among vertebrate species, and IGF-I-like molecules are found in species whose origins extend back as much as 550 million years. The insulin receptor is also highly conserved in vertebrate species, and an insulin-receptor-like molecule has been characterized in Drosophila. In contrast, IGF-I receptors have only been characterized in mammalian species and partially studied in Xenopus, in which the tyrosine kinase domain is highly conserved. Studies are presently being undertaken to analyze in more detail the regulation of the genes encoding this important family of growth factors and the structure/function relationships in the gene products themselves.

Amino Acid Sequence↗
PubMed2006

Chimeric honeybees (Apis mellifera) produced by transplantation of embryonic cells into pre-gastrula stage embryos and detection of chimerism by use of microsatellite markers.

The production of chimeras, by use of cell transplantation, has proved to be highly valuable in studies of development by providing insights into cell fate, differentiation, and developmental potential. So far, chimeric honeybees have been created by nuclear transfer technologies. We have developed protocols to produce chimeric honeybees by use of cell transplantation. Embryonic cells were transplanted between pre-gastrula stage embryos (32-34 hr after oviposition) and hatched larvae were reared in vitro for 4 days. Chimeric individuals were detected by use of microsatellite analysis and a conservative estimation approach. 4.8% of embryos, posteriorly injected with embryonic cells, developed into chimeric honeybee larvae. By injection of cells pre-stained with fluorescent cell tracer dye, we studied the integration of transplanted cells in the developing embryos. Number of injected cells varied from 0 to 50 and cells remained and multiplied mainly in the area of injection.

Animals↗
PubMed1998

Modular organization of inteins and C-terminal autocatalytic domains.

Analysis of the conserved sequence features of inteins (protein "introns") reveals that they are composed of three distinct modular domains. The N-terminal (N) and C-terminal (C) domains are predicted to perform different parts of the autocatalytic protein splicing reaction. An optional endonuclease domain (EN) is shown to correspond to different types of homing endonucleases in different inteins. The N domain contains motifs predicted to catalyze the first steps of protein splicing, leading to the cleavage of the intein N terminus from its protein host. Intein N domain motifs are also found in C-terminal autocatalytic domains (CADs) present in hedgehog and other protein families. Specific residues in the N domain of intein and CADs are proposed to form a charge relay system involved in cleaving their N-termini. The intein C domain is apparently unique to inteins and contains motifs that catalyze the final protein splicing steps: ligation of the intein flanks and cleavage of its C terminus to release the free intein and spliced host protein. All intein EN domains known thus far have dodecapeptide (DOD, LAGLI-DADG) type homing endonuclease motifs. This work identifies an EN domain with an HNH homing-endonuclease motif and two new small inteins with no EN domains. One of these small inteins might be inactive or a "pseudo intein." The results suggest a modular architecture for inteins, clarify their origin and relationship to other protein families, and extend recent experimental findings on the functional roles of intein N, C, and EN motifs.

Amino Acid Sequence↗
PubMed1996

Genomic structure of the human bradykinin B1 receptor gene and preliminary characterization of its regulatory regions.

We report on the isolation of the human BK B1 receptor gene from a human lung fibroblast genomic library. The gene is present as a single copy, spans more than 10 kilobases and includes three exons interrupted by two introns. The relative position of the exons was mapped using RNase protection assay, and genomic DNA and cDNA sequence matches. Exon and intron-exon boundaries, and 5', 3' flanking regions were sequenced. While the 5' untranslated region is distributed on all three exons, the coding region is located entirely on the third exon. The exon-intron junction sequences are highly conserved. Primer extension analysis mapped the transcriptional initiation site 21 base pairs downstream of a TATA sequence. Two Aluc sequences and two distinct functional promoters were found in the human BK B1 receptor gene.

Base Sequence↗
PubMed1996

cDNA cloning and expression of the Xenopus laevis vitellogenin receptor.

A Xenopus laevis oocyte cDNA library was screened with a PCR-generated X. laevis vitellogenin (VTG) receptor probe and a 3.6 kb cDNA clone containing the entire open reading frame, and 5' and 3' noncoding regions were isolated. The deduced amino acid sequence was 72% homologous to the chicken VLDL/VTG receptor, and the characteristic domains were highly conserved. Ligand binding analysis confirmed that the cloned receptor was Xenopus VTG-specific. Although Northern blotting analysis revealed that this gene was expressed as a major transcript of 3.6 kb in Xenopus ovary, weak but significant expression was observed in other tissues by RT-PCR analysis. The fact that major expression of the gene occurs in the ovary suggests that it has an important function in this organ.

Amino Acid Sequence↗
PubMed1999

Cloning, sequencing, and developmental expression levels of a novel glutamate-gated chloride channel homologue in the parasitic nematode Haemonchus contortus.

Glutamate-gated chloride channels (GluCls) are the proposed site of action for macrocyclic lactone anthelminthics such as ivermectin (IVM) and the milbemycins such as moxidectin (MOX). The importance of this interaction between macrocyclic lactones and GluCls is strengthened by the recent genetic evidence that GluCls are involved in IVM and MOX resistance in the parasitic nematode Haemonchus contortus (1). We have cloned two full length GluCl putative alpha-subunit cDNAs from H. contortus (HcGluCla and b) that exhibit different sized ligand binding domains. Phylogenetic analysis of the conserved regions of the amino acid sequence of HcGluCla suggests that it is a member of the GluCl family but forms a distinct subbranch within this family. The expression level of HcGluCla was examined in different developmental stages of H. contortus (eggs, L3, and adults) and found to be significantly downregulated in eggs compared to adults.

Alternative Splicing↗
PubMed2000

Molecular cloning of a novel form (two-repeat) protein related to voltage-gated sodium and calcium channels.

Voltage-gated Ca and Na channels share similar structure: four homologous domains (I-IV), each with six transmembrane segments (S1-S6). They may be formed by two rounds of duplication of a single channel domain similar to voltage-activated potassium channels. However, the channels with the intermediate structure, namely, two-domain channels have not yet been identified. We report here the cloning of a novel protein from rat kidney that contains two domains (I-II), each with S1-S6 segments that are found in voltage-gated Ca and Na channels. Because of unusual structure, the protein was named two-pore channel 1 (TPC1). TPC1 encodes 819 amino acids with two conserved positively charged voltage sensor segments (S4) but the pore segments are not conserved. Northern blot analysis showed that TPC1 mRNA (5 kb) was expressed widely. It was expressed at relatively high level in kidney, liver, and lung. Immunohistochemistry of kidney revealed that TPC1 was expressed at inner medullary collecting ducts. In expression studies, no functional currents could be detected in CHO cells and Xenopus oocytes. Based on its primary structure, we propose that TPC1 might be a predecessor of the conventional four repeat voltage-gated Ca and Na channels and will give insights into the evolution of ion channels.

Amino Acid Sequence↗
PubMed2000

Activation of fgf4 gene expression in the myotomes is regulated by myogenic bHLH factors and by sonic hedgehog.

The Fgf4 gene encodes an important signaling molecule which is expressed in specific developmental stages, including the inner cell mass of the blastocyst, the myotomes, and the limb bud apical ectodermal ridge (AER). Using a transgenic approach, we previously identified overlapping but distinct enhancer elements in the Fgf4 3' untranslated region necessary and sufficient for myotome and AER expression. Here we have investigated the hypothesis that Fgf4 is a target of myogenic bHLH factors. We show by mutational analysis that a conserved E box located in the Fgf4 myotome enhancer is required for Fgf4-lacZ expression in the myotomes. A DNA probe containing the E box binds MYF5, MYOD, and bHLH-like activities from nuclear extracts of differentiating C2-7 myoblast cells, and both MYF5 and MYOD can activate gene expression of reporter plasmids containing the E-box element. Analyses of Myf5 and MyoD knockout mice harboring Fgf4-lacZ transgenes show that Myf5 is required for Fgf4 expression in the myotomes, while MyoD is not, but MyoD can sustain Fgf4 expression in the ventral myotomes in the absence of Myf5. Sonic hedgehog (Shh) signaling has been shown to have an essential inductive function in the expression of Myf5 and MyoD in the epaxial myotomes, but not in the hypaxial myotomes. We show here that expression of an Fgf4-lacZ transgene in Shh-/- embryos is suppressed not only in the epaxial but also in the hypaxial myotomes, while it is maintained in the AER. This suggests that Shh mediates Fgf4 activation in the myotomes through mechanisms independent of its role in the activation of myogenic factors. Thus, a cascade of events, involving Shh and bHLH factors, is responsible for activating Fgf4 expression in the myotomes in a spatial- and temporal-specific manner.

3' Untranslated Regions↗