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PubMed1999

Identification of soluble interleukin-4 receptor in rat glomerular epithelial cells(1).

Interleukin (IL)-4, a pleiotropic cytokine involved in many glomerular diseases, is regulated positively by membrane-bound IL-4R (mIL-4R) and negatively by soluble IL-4R (sIL-4R). Because natural sIL-4R has been documented only in mice, we undertook this study in rats to determine whether they, too, express sIL-4R, particularly in kidney cells. A pair of IL-4R primers was designed for this purpose and used in the polymerase chain reaction. As a result, sIL-4R was found not only in rats spleen cells but also in their glomerular epithelial cells (GEC). Sequence analysis revealed that the mRNA of rat sIL-4R has a 75-bp insert sequence. This insert generated a termination TGA codon upstream from the transmembrane region, resulting in formation of the sIL-4R. Subsequent screening of the kidney cDNA library enabled us to obtain the whole 3605-bp cDNA of sIL-4R; the full-length 3530-bp mIL-4R cDNA was also identified as a much longer sequence than previously published. Among the total 39 clones positive for IL-4R, two were confirmed as sIL-4R, and 37 clones were positive for mIL-4R. Next, the translated portion of sIL-4R cDNA was constructed into an expression vector, enabling us to obtain a recombinant sIL4R-myc fusion protein. By using this recombinant sIL-4R, we proved that sIL-4R can antagonize the IL-4-induced proliferation of spleen cells. Present study demonstrated that sIL-4R is expressed in kidney cells and antagonistically functional.

Animals↗
PubMed2000

Protein structure computing in the genomic era.

Functional analysis of the proteins discovered in fully sequenced genomes represents the next major challenge of life science research. Computational methods play a crucial role in this activity and, among them, comparative protein modelling is of great assistance during the rational design of mutagenesis experiments. Our aim over the last several years has been to further the use of 3-D model structures in this field. Therefore, we have developed a comparative protein modelling environment composed of the Swiss-PdbViewer (sequence to structure workbench and viewing program), SWISS-MODEL (internet-based server for model generation) and a database of a model generated with 3DCrunch.

Computational Biology↗
PubMed2005

CTCF binding and higher order chromatin structure of the H19 locus are maintained in mitotic chromatin.

Most of the transcription factors, RNA polymerases and enhancer binding factors are absent from condensed mitotic chromosomes. In contrast, epigenetic marks of active and inactive genes somehow survive mitosis, since the activity status from one cell generation to the next is maintained. For the zinc-finger protein CTCF, a role in interpreting and propagating epigenetic states and in separating expression domains has been documented. To test whether such a domain structure is preserved during mitosis, we examined whether CTCF is bound to mitotic chromatin. Here we show that in contrast to other zinc-finger proteins, CTCF indeed is bound to mitotic chromosomes. Mitotic binding is mediated by a portion of the zinc-finger DNA binding domain and involves sequence specific binding to target sites. Furthermore, the chromatin loop organized by the CTCF-bound, differentially methylated region at the Igf2/H19 locus can be detected in mitosis. In contrast, the enhancer/promoter loop of the same locus is lost in mitosis. This may provide a novel form of epigenetic memory during cell division.

Animals↗
PubMed1993

Synchrony of cell spreading and contraction force as phagocytes engulf large pathogens.

A simple micromechanical method has been used to directly measure the force of contraction in single mammalian phagocytes (blood granulocytes) during engulfment of large yeast pathogens. Both the time course of cell spreading over the yeast particle and increase in cell body contractile force were quantitated at three temperatures in the range of 23-35 degrees C. The surprising feature of the phagocyte response was that engulfment and cell body contraction occurred in a serial sequence: i.e., the phagocyte spread rapidly over the particle at a steady rate with no detectable cell body contraction; when spreading stopped, contraction force in the cell body then rose steadily to a plateau level that remained stationary until the next sequence of spreading and contraction. Both spreading and contraction exhibited abrupt start/stop kinetics. Also impressive, the cell contraction force stimulated by phagocytosis was quite large (approximately 10(-8) N)-two orders of magnitude larger than the force necessary to deform passive phagocytes to the same extent. If distributed uniformly over the cell cross section, the contraction force is equivalent to an average contractile stress of approximately 10(3) N/m2 (0.01 Atm). These physical measurements in situ set critical requirements for the mechanism of force generation in granulocytes, imply that a major increase in network cross-linking accompanies build-up in contractile force and that subsequent network dissolution is necessary for locomotion.

Biomechanical Phenomena↗
PubMed1992

Mitochondrial DNA reveals formation of nonhybrid frogs by natural matings between hemiclonal hybrids.

The European water frog Rana esculenta (RL), a natural hybrid between R. ridibunda (RR) and R. lessonae (LL), reproduces by hybridogenesis: haploid gametes usually contain an intact chromosome set of R. ridibunda (R); the lessonae nuclear genome (L) is lost from the germ line. Hybridity is restored in the next generation, via fertilization by syntopic R. lessonae. Matings between two hybrids (RL x RL) usually give inviable R. ridibunda (RR) progeny. The adult R. ridibunda subpopulation of Trubeschloo, a gravel pit in northern Switzerland, consists only of females. Fragment patterns for mitochondrial DNA (mtDNA) of these R. ridibunda were identical with those of syntopic R. esculenta and of local populations of R. lessonae; they differed from the patterns in eastern European populations of R. lessonae and of R. ridibunda mtDNAs (3.7% and 9.3% estimated sequence divergence, respectively). In contrast, mtDNAs of two R. ridibunda from an introduced Swiss population with both sexes, although different (2.7% divergence) from each other, were typical R. ridibunda rather than R. lessonae mtDNAs. These data, together with unisexuality, demonstrate conclusively that the all-female R. ridibunda population at Trubeschloo originated from matings between two R. esculenta. The formation of independently reproducing R. ridibunda populations via such hybrid x hybrid matings is precluded because progeny of these matings are unisexual. Recombination in the regenerated fertile R. ridibunda females, followed by matings with R. lessonae, nevertheless provides a mechanism for meiotic reshuffling of genetic material in ridibunda haplotypes that is not typically available in hemiclonal lineages.

Animals↗
PubMed1995

Linkage analysis of the gene encoding precursor protein of diapause hormone and pheromone biosynthesis-activating neuropeptide in the silkmoth, Bombyx mori.

We have determined the map position of the gene encoding a common precursor protein for diapause hormone and pheromone biosynthesis-activating neuropeptide (the DH-PBAN gene, Dh) in the silkmoth, Bombyx mori. First we compared the structure of introns in the DH-PBAN gene by the polymerase chain reaction, and found that the Dh locus carried three alleles, DhA1, DhA2 and DhB. The DhA1 and DhA2 alleles contained a fourth intron consisting of 740 bp, whereas DhB had a longer fourth intron of 770 bp. DhA1 and DhA2 contained a fifth intron consisting of 940 bp, whereas the fifth intron in DhB was much longer and consisted of 1700 bp. DhA1 was distinguished from DhA2 by an RFLP in the fifth intron after digestion with Rsa I. Linkage analyses using these polymorphisms showed that Dh was linked to the bp gene on chromosome 11, and independent of markers on chromosomes 1, 2, 3, 4, 5, 6, 7 and 13. To determine the map position, we obtained F1 hybrids between the n501 strain (K DhA1) and the w30 strain (+K DhB), and backcrossed the F1 hybrid to females of the w30 strain. From the segregation of K and Dh in 864 individuals in the next generation, the recombination value was calculated as 25.5% between K and Dh. Similarly we obtained backcross progeny between the No. 744 strain (Bu DhA1) and the w30 strain (+Bu DhB), and calculated the recombination value between Bu and Dh as 30.4% from 487 progeny.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.

Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.

Amino Acid Sequence↗
PubMed2003

Membrane type-1 matrix metalloproteinase functions as a proprotein self-convertase. Expression of the latent zymogen in Pichia pastoris, autolytic activation, and the peptide sequence of the cleavage forms.

An understanding of the regulatory mechanisms that control the activity of membrane type-1 matrix metalloproteinase (MT1-MMP), a key proteinase in tumor cell invasion, is essential for the design of potent and safe anti-cancer therapies. A unique proteolytic pathway regulates MT1-MMP at cancer cell surfaces. The abundance of proteolytic enzymes in cancer cells makes it difficult to identify the autocatalytic events in this pathway. To identify these events, a soluble form of MT1-MMP, lacking the C-terminal transmembrane and cytoplasmic domains, was expressed in Pichia pastoris. Following secretion, the latent zymogen and active enzyme were each purified from media by fast protein liquid chromatography. Trace amounts of active MT1-MMP induced activation of the zymogen and its self-proteolysis. This autocatalytic processing generated six main forms of MT1-MMP, each of which was subjected to the N-terminal microsequencing to identify the cleavage sites. Our data indicate that MT1-MMP functions as a self-convertase and is capable of cleaving its own prodomain at the furin cleavage motif RRKR downward arrow Y(112), thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr(112). The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp(119) and at Asn(130)) and, next, to the three inactive ectodomain forms (N terminus at Thr(222), at Gly(284), and at Thr(299)). These findings provide, for the first time, a structural basis for understanding the unconventional mechanisms of MT1-MMP activation and regulation. Finally, our data strongly imply that MT1-MMP is a likely substitute for the general proprotein convertase activity of furin-like proteinases, especially in furin-deficient cancer cells.

Amino Acid Sequence↗
PubMed2002

An automated two-dimensional optical force clamp for single molecule studies.

We constructed a next-generation optical trapping instrument to study the motility of single motor proteins, such as kinesin moving along a microtubule. The instrument can be operated as a two-dimensional force clamp, applying loads of fixed magnitude and direction to motor-coated microscopic beads moving in vitro. Flexibility and automation in experimental design are achieved by computer control of both the trap position, via acousto-optic deflectors, and the sample position, using a three-dimensional piezo stage. Each measurement is preceded by an initialization sequence, which includes adjustment of bead height relative to the coverslip using a variant of optical force microscopy (to +/-4 nm), a two-dimensional raster scan to calibrate position detector response, and adjustment of bead lateral position relative to the microtubule substrate (to +/-3 nm). During motor-driven movement, both the trap and stage are moved dynamically to apply constant force while keeping the trapped bead within the calibrated range of the detector. We present details of force clamp operation and preliminary data showing kinesin motor movement subject to diagonal and forward loads.

Automation↗
PubMed2000

Delayed activation of post-transcriptional gene silencing and de novo transgene methylation in plants with the coat protein gene of sweet potato feathery mottle potyvirus.

The relationship between post-transcriptional gene silencing (PTGS) and DNA methylation was examined using Nicotiana benthamiana transformed with the coat protein gene including the 3' non-translated region of sweet potato feathery mottle potyvirus. Line 4.28 showed a delayed activation of the transgene silencing in comparison with the other silenced lines, and showed complete resistance against the recombinant potato virus X engineered to contain the sequence homologous to the transgene when the silencing was activated. The transgene methylation in line 4.28 was less extensive in comparison with those of the other silenced lines before the silencing was activated. However, the extent of methylation increased in the course of plant development and became comparable with those in the other silenced lines. The activated silencing and the increased transgene methylation were reset after meiosis. However, the characters of delayed activation of the silencing and developmentally increased transgene methylation were meiotically transmitted to the next generation. These results suggest that transgene(s) itself has a potential to trigger and reset DNA methylation, which could determine a state of PTGS.

Journal Article↗
PubMed1979

Some sequence similarities among cloned mouse DNA segments that code for lambda and kappa light chains of immunoglobulins.

A comparison between the cloned mouse DNA segments that were found to code for the lambda and kappa light chains of immunoglobulins established that there were seven short nucleotide sequences, two of which matched 6 out of 7, two 7 out of 8, two 8 out of 9, and one 9 out of 10 bases; these sequences were located either at homologous amino acid positions or at positions displaced by four amino acids or less. They all occurred in the framework regions (FRs), five next to the complementarity-determining regions (CDRs). Three of these were unique and did not occur elsewhere in the immunoglobulin nucleotides sequenced thus far or in DNA's of phage phi X174, phage G4, or simian virus 40. Five could serve as sites of joining by recombination or insertion of CDR to FR segments, and the invariant tryptophan that is the first residue of the second FR might serve as a sixth. These sites are consistent with the mini-gene or insertional hypotheses for the generation of antibody diversity but could also serve as points of recognition for a mutator enzyme or could serve to limit somatic mutation to the CDRs.

Amino Acid Sequence↗
PubMed2001

A novel transposon tagging element for obtaining gain-of-function mutants based on a self-stabilizing Ac derivative.

A novel tagging system AcREH, designed for obtaining gain-of-function mutations, was prepared on the basis of a self-stabilizing Ac transposon derivative. The transposable element, DsAT, was constructed in a way that it can activate transcription of neighboring genes by two 35S promoters and/or by four tandem repeats of the enhancer fragment of this promoter. DsAT revealed somatic excision in the first generation of the tobacco transformants. The element exhibited germinal excision to the next generation, as demonstrated by PCR and Southern hybridization analysis. In spite of the structure of the element, which may inhibit the expression of the transposase gene, the frequency of germinal excision was comparable to or higher than those so far reported, suggesting the applicability of the element for gene tagging.

Base Sequence↗
PubMed1992

Sequence and in vitro expression of the M2 gene of turkey rhinotracheitis pneumovirus.

Negative-stranded virion RNA and oligonucleotide primers complementary to fusion (F) protein gene sequences were used to generate cDNA clones, revealing that the gene 5'-proximal to the F protein corresponded to the M2 (22K) gene, as in respiratory syncytial (RS) virus. The transcription start signal, GGGACAAGU, was identical to that of the F and matrix (M) proteins of turkey rhinotracheitis virus (TRTV). There were two sequences with the potential to function as transcription termination/poly(A) signals, located at nucleotides 751 to 762 and 777 to 787; 15 clones derived from mRNA indicated that the first of these sequences formed the major signal. Part of the next downstream (5') gene was sequenced; unlike mammalian pneumoviruses the TRTV M2 gene did not overlap the beginning of the 5'-proximal gene. Northern blotting indicated that infected Vero cells contained less M2 mRNA than F mRNA and that about half of the M2 mRNA was present as a F-M2 dicistronic mRNA. The M2 gene contained two overlapping open reading frames (ORFs 1 and 2), as with RS virus. ORF 1 comprised 558 nucleotides with the coding potential for a 186 amino acid polypeptide, M(r) 20959, eight or nine residues shorter than for human RS virus strains. The overall amino acid identity was 40%, the N-terminal one-third of the proteins sharing 62% of residues, the remainder 29%. A hydropathy plot of the TRTV M2 protein had close similarity to that of the M2 or RS virus. The protein was predicted to have a basic character with no N-terminal signal sequence or other major highly hydrophobic sequences. In vitro translation of a transcript comprising both ORFs 1 and 2 produced a single product of apparent M(r) 23000, corresponding to the M2 product of ORF 1. Site-directed mutagenesis confirmed that this product was derived from ORF 1 and that frameshifting was not involved. The second ORF was expressed only from a transcript which lacked the AUG codons of ORF 1 and, although occupying a similar position to that in the RS virus M2 gene, had virtually no amino acid identity in its 73 residue length and was approximately 25% shorter than the corresponding RS virus ORF 2. The hydropathy plot of the potential products of the second ORFs of TRTV and RS virus showed little resemblance. Taken together these results suggest that ORF 2 is unlikely to be expressed in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2002

A simple model of long-term spike train regularization.

A simple model of spike generation is described that gives rise to negative correlations in the interspike interval (ISI) sequence and leads to long-term spike train regularization. This regularization can be seen by examining the variance of the kth-order interval distribution for large k (the times between spike i and spike i + k). The variance is much smaller than would be expected if successive ISIs were uncorrelated. Such regularizing effects have been observed in the spike trains of electrosensory afferent nerve fibers and can lead to dramatic improvement in the detectability of weak signals encoded in the spike train data (Ratnam & Nelson, 2000). Here, we present a simple neural model in which negative ISI correlations and long-term spike train regularization arise from refractory effects associated with a dynamic spike threshold. Our model is derived from a more detailed model of electrosensory afferent dynamics developed recently by other investigators (Chacron, Longtin, St.-Hilaire, & Maler, 2000;Chacron, Longtin, & Maler, 2001). The core of this model is a dynamic spike threshold that is transiently elevated following a spike and subsequently decays until the next spike is generated. Here, we present a simplified version-the linear adaptive threshold model-that contains a single state variable and three free parameters that control the mean and coefficient of variation of the spontaneous ISI distribution and the frequency characteristics of the driven response. We show that refractory effects associated with the dynamic threshold lead to regularization of the spike train on long timescales. Furthermore, we show that this regularization enhances the detectability of weak signals encoded by the linear adaptive threshold model. Although inspired by properties of electrosensory afferent nerve fibers, such regularizing effects may play an important role in other neural systems where weak signals must be reliably detected in noisy spike trains. When modeling a neuronal system that exhibits this type of ISI correlation structure, the linear adaptive threshold model may provide a more appropriate starting point than conventional renewal process models that lack long-term regularizing effects.

Action Potentials↗
PubMed2001

Transposition and gene disruption in the male germline of the mouse.

We have tested a synthetic, functional, transposon called Sleeping Beauty for use in mice as a germline insertional mutagen. We describe experiments in which mutagenic, polyadenylation-site trapping, transposon vectors were introduced into the germline of mice. When doubly transgenic males, expressing the Sleeping Beauty transposase gene (SB10) and harboring poly(A)-trap transposon vectors, were outcrossed to wild-type females, offspring were generated with new transposon insertions. The frequency of new transposon insertion is roughly two per male gamete. These new insertions can be passed through the germline to the next generation and can insert into or near genes. We have generated a preliminary library of 24 mice harboring 56 novel insertion sites, including one insertion into a gene represented in the EST database and one in the promoter of the galactokinase (Gck) gene. This technique has promise as a new strategy for forward genetic screens in the mouse or functional genomics.

Animals↗
PubMed1992

Structure and early embryonic expression of the zebrafish engrailed-2 gene.

The Drosophila homeobox gene engrailed (en) is needed for correct embryonic development, and related sequences are active during vertebrate embryogenesis. Here we report the protein coding sequence and embryonic expression pattern of the zebrafish engrailed-2 gene (eng-2) which is directly homologous to En-2 in mice and Xenopus. The predicted zebrafish Eng-2 protein shares 65% overall identity to its Xenopus counterpart. In addition to the highly conserved homeodomain region, sequence conservation is present within three short stretches in the N-terminal region. The embryonic expression of the eng-2 gene was analysed by in situ hybridization to whole-mount embryos and tissue sections. Transcripts are first detected in two lateral bands at the 10-h stage, when epiboly is completed. Within the next 2 h of development, these two bands migrate and fuse at the midline. By the time the neural keel becomes visible (11-12 h), a transverse stripe of eng-2 expressing cells is seen at the presumptive midbrain-hindbrain boundary. Later this stripe becomes significantly compressed along the AP axis, and in 24-h embryos eng-2 transcripts are detected mainly in the posterior midbrain. In the hindbrain, eng-2 expression seems restricted to the primordium of the cerebellum. A second site of activity was observed in each somite where specific myotomal cells, the muscle pioneers, express eng-2. Our observations are discussed in relation to early regionalization of the central nervous system (CNS) and the generation of morphological borders.

Amino Acid Sequence↗
PubMed2026

A novel allele of Sh1 facilitates the development of waxy-sweet corn from waxy corn.

Waxy corn and sweet corn represent 2 major classes of fresh-eating corn, each with distinct sensory attributes and nutritional compositions. Developing a new variety that combines both waxy and sweet traits would address rising consumer demand and expand new market potential. From a fast neutron-mutagenized population of the waxy corn inbred line HB522, we isolated a novel mutant, designated as wx-sweet, whose kernels simultaneously exhibit waxy and sweet characteristics at the milk-filling stage. Through bulked segregant analysis combined with fine mapping, we mapped the causal locus to SHRUNKEN1 (Sh1) on chromosome 9, which was confirmed by an allelism test with a characterized Mu-insertion allele of Sh1. A 7,227-bp Copia-type long terminal repeat retrotransposon insertion was identified in exon 2 of Sh1 in the wx-sweet mutant by long-read sequencing. Consistently, the novel sh1 allele significantly reduced sucrose synthase activity. Genetic and physiological analyses demonstrate that sh1 and wx1 act synergistically to fine-tune carbohydrate metabolism in the endosperm. Integrated transcriptomic and metabolomic profiling uncover extensive transcriptional reprogramming and redirected metabolic flux, leading to substantial accumulation of sucrose and a range of oligosaccharides. These metabolic shifts underlie the unique simultaneous dual waxy-sweet texture in fresh-eating wx-sweet kernels. In summary, our work not only provides valuable genetic resources for breeding next-generation fresh-eating corn but also, for the first time, elucidates the molecular mechanism by which the sh1 and wx1 mutations cooperatively shape the waxy-sweet endosperm phenotype.

Zea mays↗
PubMed1999

MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.

The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.

Animals↗