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PubMed2004

Single-molecule fluorescence spectroscopy and microscopy of biomolecular motors.

The methods of single-molecule fluorescence spectroscopy and microscopy have been recently utilized to explore the mechanism of action of several members of the kinesin and myosin biomolecular motor protein families. Whereas ensemble averaging is removed in single-molecule studies, heterogeneity in the behavior of individual motors can be directly observed, without synchronization. Observation of translocation by individual copies of motor proteins allows analysis of step size, rate, pausing, and other statistical properties of the process. Polarization microscopy as a function of nucleotide state has been particularly useful in revealing new and highly rotationally mobile forms of particular motors. These experiments complement X-ray and biochemical studies and provide a detailed view into the local dynamical behavior of motor proteins.

Fluorescence Polarization↗
PubMed2005

HeT-A and TART, two Drosophila retrotransposons with a bona fide role in chromosome structure for more than 60 million years.

Drosophila telomeres have been maintained by retrotransposition for at least 60 MY, which predates the separation of extant species of this genus. Studies of D. melanogaster, D. yakuba, and D. virilis show that, in Drosophila, telomeres are composed of two non-LTR retrotransposons, HeT-A and TART. Far from being static, HeT-A and TART evolve faster than Drosophila euchromatic genes. In spite of their high rate of sequence change, HeT-A and TART maintain their basic structures and unusual individual features. The maintenance of their separate identities suggests that HeT-A and TART cooperate either in the process of retrotransposition onto the chromosome end, or in the formation of telomere chromatin by transposed DNA copies. The telomeric retrotransposons and the Drosophila genome constitute an example of a robust symbiotic relationship between mobile elements and the genome.

Animals↗
PubMed1993

In humans all U3 genes map to chromosome 17p12-->p11, but in mouse the U3A and U3B genes are located on different chromosomes.

U3 small nucleolar RNA, which participates in eukaryotic rRNA processing, is encoded by a small multigene family in mammals. In humans, the four to six gene copies code for an identical U3 RNA molecule; rodents, however, have two variant forms of these genes, U3A and U3B. We show that all U3 genes in humans map to a single chromosomal locus, 17p12-->-p11, which corresponds exactly to the region of mouse Chromosome 11 where the four U3B genes are clustered. By contrast, in mouse the unique U3A gene copy is not linked to the U3B gene cluster but maps to another chromosome (the B2-B4 region of Chromosome 10).

Animals↗
PubMed1993

Haemophilia B due to a de novo insertion of a human-specific Alu subfamily member within the coding region of the factor IX gene.

A de novo insertion of an Alu repeated DNA element was found within exon V of the factor IX gene in a patient with severe haemophilia B. The element interrupts the reading frame of the mature factor IX at glutamic acid 96 resulting in a stop codon within the inserted sequence. The Alu repeat is 322 bp long, and the 5' region is shortened by 38 bp. The insertion created a target site duplication of 15 bp consistent with retroposition, and contains a pure polyadenine tract of at least 78 resides at the 3' end. The nucleotide sequence agrees with a consensus for an Alu subfamily which is evolutionarily the most recently inserted, suggesting that it is an exact copy of a putative source gene. These observations indicate that retroposition of Alu elements is a continual process and a mechanism for generating human genetic defects.

Base Sequence↗
PubMed2003

A computational model as neurodecoder based on synchronous oscillation in the visual cortex.

Based on synchronized responses of neuronal populations in the visual cortex to external stimuli, we proposed a computational model consisting primarily of a neuronal phase-locked loop (NPLL) and multiscaled operator. The former reveals the function of synchronous oscillations in the visual cortex. Regardless of which of these patterns of the spike trains may be an average firing-rate code, a spike-timing code, or a rate-time code, the NPLL can decode original visual information from neuronal spike trains modulated with patterns of external stimuli, because a voltage-controlled oscillator (VCO), which is included in the NPLL, can precisely track neuronal spike trains and instantaneous variations, that is, VCO can make a copy of an external stimulus pattern. The latter, however, describes multi-scaled properties of visual information processing, but not merely edge and contour detection. In this study, in which we combined NPLL with a multiscaled operator and maximum likelihood estimation, we proved that the model, as a neurodecoder, implements optimum algorithm decoding visual information from neuronal spike trains at the system level. At the same time, the model also obtains increasingly important supports, which come from a series of experimental results of neurobiology on stimulus-specific neuronal oscillations or synchronized responses of the neuronal population in the visual cortex. In addition, the problem of how to describe visual acuity and multiresolution of vision by wavelet transform is also discussed. The results indicate that the model provides a deeper understanding of the role of synchronized responses in decoding visual information.

Action Potentials↗
PubMed2002

Characterization of the AeaHP gene and its expression in the mosquito Aedes aegypti (Diptera: Culicidae).

The sequence and tissue expression of the gene encoding a peptide hormone Aea-HP-I, known to inhibit host-seeking behavior, has been characterized for the yellowfever mosquito, Aedes aegypti (L.). The open reading frame reveals a prepropeptide that would be processed into three identical peptides. The gene contains four short introns and exists as a single genomic copy. Transcripts of the gene were present in the brain, terminal ganglion, and midgut of adults, and in females, its expression profile differed for each tissue before and during a reproductive cycle. Peptides resulting from this expression were identified in the female tissues by immunoassays. Numerous neurosecretory cells and neurons in the nervous system were immunostained by an Aea-HP-I antiserum. Hundreds of endocrine cells were stained similarly in the midgut, thus contributing to the 10 times greater amount of immunoreactive peptide in an abdomen than in a head, as determined with an Aea-HP-I radioimmunoassay. Based on these results, neurosecretory cells and midgut endocrine cells are likely sources of Aea-HPs shown to reach highest hemolymph titer at the same time as host seeking is inhibited in female Ae. aegypti during a reproductive cycle.

Aedes↗
PubMedDate not supplied

Stability of a dry laser imaging system.

Laser imagers are the method of choice for creating hard copies of electronic images, such as computed tomograms and computed radiographs. New dry laser imagers use heat processing rather than wet chemical processing. This article describes tests assessing the stability and reproducibility of dry-processed laser images under normal conditions. The authors conclude that dry laser images compare favorably with wet laser images, although testing under extreme conditions is needed.

Image Processing, Computer-Assisted↗
PubMed1991

Abundance and stability of erythropoietin receptor mRNA in mouse erythroid progenitor cells.

The abundance and stability of erythropoietin (EPO) receptor mRNA was measured in Friend virus-infected mouse splenic erythroblast (FVA) cells. FVA cells correspond to the colony-forming unit-erythroid (CFU-E) and cluster-forming unit-erythroid stages of differentiation and they mature in vitro into reticulocytes during 45 to 60 hours of culture. After 20 hours, there was a rapid decline in the EPO receptor mRNA level through the 45-hour culture period. Levels of actin and beta-globin mRNAs were monitored as two representative proteins that are actively synthesized in early versus late stages of terminal erythroid differentiation. A general decrease of actin mRNA level was apparent, whereas globin mRNA levels increased throughout the culture period. The greatest decrease in both EPO receptor and actin mRNA was observed when the cell population was at the stage of basophilic and polychromatophilic erythroblasts. The half-life for EPO receptor mRNA was approximately 75 minutes, as determined using the transcriptional inhibitor actinomycin D. The half-life measured at several times during the 48-hour culture period remained constant. These results indicate that EPO receptor mRNA must be transcribed continuously until late in the maturation process of FVA cells in order to maintain the levels seen by Northern analysis. The number of copies of EPO receptor mRNA at 0 hours was determined to be 25 copies per cell. This low number and the decline of EPO receptor mRNA correlate with the low receptor numbers on FVA cells, as well as the decline of binding sites with cell maturation.

Actins↗
PubMed1991

Cytochrome c oxidase subunit VIIa isoforms. Characterization and expression of bovine cDNAs.

Subunit VIIa of bovine cytochrome c oxidase occurs in two forms, the so-called heart and liver isoforms, which have been shown by protein analysis to differ in 35% of their amino acids. We have isolated and characterized cDNAs for each isoform. The derived heart-type processed protein is 59 amino acids long, with a 21-residue presequence; the immediate C terminus differs from the established protein sequence. The liver-type processed protein is 60 residues long, with a 23-amino acid presequence. Both presequences are traditional in that they are positively charged and appear amphiphilic when helically arrayed. The presequences are only 22% identical, but they both contain conserved residues indicative of two-step processing of the precursor proteins. Southern blot analysis reveals that the bovine genome contains five to six copies of the liver-type gene as opposed to the presence of a single copy heart-type gene. Transcriptional analysis shows that heart-type message is detectable only in heart and skeletal muscle; the liver type is also seen in heart and muscle and, additionally, in the other tissues examined (liver, brain, and lung). The amino acid sequence EKQKLFQED is conserved in rat and in both isoforms in cow and human and may represent a domain of core subunit function.

Amino Acid Sequence↗
PubMed1989

The nature of X-ray and chemically induced mutations in Drosophila in relation with DNA repair.

This paper describes the spectrum of mutations induced by alkylating agents and ionizing radiation in Drosophila. Specifically, the genotoxic profile of the alkylating agents is set against their carcinogenic potency. Alkylating agents that react preferentially with N-atoms in the DNA are relatively poor mutagens, especially so in repair-competent (early) germ cells, and likewise weak carcinogens when compared to those that are more efficient in O-alkylation. Genetic techniques combined with molecular analysis of X-ray and neutron induced mutations show that ionizing radiation induces primarily break-type mutations in a repair proficient background. Both multi-locus deletions as well as small intragenic deletions of only a few base-pairs are observed. The small deletions occur between direct repeats of 2-3 nucleotides, one copy of which is retained in the mutant allele. Possibly, these deletions are the result of repair processes. The effect of changes in DNA-repair (excision repair deficient) is reflected by a "hypermutability" for alkylating agents specifically for N-alkylators, indicating that the normal efficient error-free repair of N-alkylation damage can explain the high exposure doses required for tumor induction in mammals. The frequency of X-ray induced whole-body white mutations, recovered in excision repair deficient Drosophila, is only slightly enhanced, when compared to the repair proficient situation. In contrast, mosaic mutations occur 3-4 times more frequent, indicating that part of the X-ray damage, normally removed by the excision repair process, is not a major impedement during replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗
PubMed1994

Gene expression in Frankia: characterization of promoters.

Promoter regions harbouring typical -10 and -35 sites of prokaryotes were isolated from the genome of various species of Frankia using an Escherichia coli promoter-probe plasmid. These promoter regions initiated transcription in E. coli and Streptomyces cells. The consensus promoter sequence for the -10-like site was T A G/A G G/A T and for the -35-like site was T T G T/A C G. Despite the GC pressure observed in Frankia, functionally important positions in the promoter are conserved. As a consequence of these studies, a marker gene having properties compatible with the transcriptional and translational processes of Frankia was built. It is an npt-II gene under the control of three copies of one of the Frankia-E. coli type promoters studied.

Actinomycetales↗
PubMed1976

[A coordinate and area recording microscope for the topographic analysis of particle size distributions (author's transl)].

A coordinate recording microscope equipped with a rolling disc planimeter is described. The application of the instrument for the analysis of cell size distribution in histologic preparations is demonstrated. The microscope stage is moved in x, and y directions by two digital micrometer spindles. The planimeter is equipped with a rotary encoder. The spindles and the rotary encoder are connected to digital counters which are input to a multiplexer. The multiplexer outputs the data to a teletype terminal both as hard copy and on paper tape. To avoid repeated measurements a video-acoustic monitoring system is provided. Processing of the data is carried out off-line with a IBM 1130 computer. Here the measured cells are displayed as points or circles on an oscilloscope. The distribution of cells of any size can be studied topographically in any region of the field of measurements.

Cells↗
PubMed2001

Self-organisation and orderly processes by individual protein complexes in the bacterial cell.

In the bacterial cell, individual multimeric proteins and multiprotein assemblies perform and control orderly processes. Individual motor enzyme complexes accomplish highly complex functions, such as nucleic acid and protein syntheses, with impressive efficiency and fidelity. Lac operon repression by the lac repressor is effectively controlled via a single molecular switch. There are only few copies of, for example, DNA polymerase holoenzyme and lac repressor and few specific target molecules/sites, with which these protein complexes interact, present in a single E. coli cell. These interactive processes take place in submicron-sized spaces characterised by extreme crowding (volume exclusion) of macromolecules and small molecules, heterogeneity and non-ideality. Recent evidence reinforces the fundamental difference of the cytoplasmic as compared with in vitro ("test tube") reaction conditions. This is reflected in the breakdown of the applicability of "bulk phase" thermodynamic, macroscopic chemical kinetic and diffusion laws to interactions of individual macromolecules and target sites in a single cell. Stochastic kinetic models and stochastic simulations enable the statistical description and analysis of biochemical reactions and binding processes which involve small numbers of reactants. New unifying concepts and models are required for the quantitative understanding of the microscopic self-organisation of multi-protein complexes and the dynamic order at the single-protein assembly and single-switch level in the living cell.

Bacteria↗
PubMed1990

The primary structure of rat ribosomal proteins: the amino acid sequences of L27a and L28 and corrections in the sequences of S4 and S12.

The amino acid sequences of rat ribosomal proteins L27a and L28 were deduced from the sequences of nucleotides in recombinant cDNAs and confirmed from the NH2-terminal amino acid sequences of the proteins. L27a contains 147 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 16 476. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 18-22 copies of the L27a gene. The mRNA for the protein is about 600 nucleotides in length. L27a is homologous to mouse L27a (there are 3 amino acid changes) and to yeast L29. Rat ribosomal protein L28 has 136 amino acids (its NH2-terminal methionine is also processed after translation) and has a molecular weight of 15 707. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 9 or 10 copies of the L28 gene. The mRNA for the protein is about 640 nucleotides in length. L28 contains a possible internal duplication of 9 residues. Corrections are recorded in the sequences reported before for rat ribosomal proteins S4 and S12.

Amino Acid Sequence↗
PubMed2003

Structure of the micronuclear alpha-tubulin gene in the phyllopharyngean ciliate Chilodonella uncinata: implications for the evolution of chromosomal processing.

Ciliates are a group of microbial eukaryotes defined by the presence of dimorphic nuclei-each cell contains both a transcriptionally active macronucleus and a germline micronucleus. During the development of the macronucleus, germline chromosomes are rearranged through extensive fragmentation, removal of internally excised sequences (IESs) and DNA amplification. We have characterized three IESs in the gene that encodes alpha-tubulin in the phyllopharyngean ciliate Chilodonella uncinata. The IESs are located within the coding domain, range in size from 81 to 107 bp, and are flanked by direct repeats that vary in length from 6 to 8 bp. All three IESs are moderately AT-rich and each contains two copies of a conserved sequence motif. These data provide evidence for the existence of IESs in phyllopharyngean ciliates and suggest that IES processing in C. uncinata may rely on a novel cis-acting sequence. Comparisons of the IESs in C. uncinata with those of 'model' ciliates-Paramecium, Tetrahymena, Euplotes, Oxytricha and Stylonychia-reveal considerable variation in chromosomal processing among ciliates.

Animals↗
PubMed1996

SOM 1, a small new gene required for mitochondrial inner membrane peptidase function in Saccharomyces cerevisiae.

IMP1 encodes a subunit of the mitochondrial inner membrane peptidase responsible for the proteolytic processing of cytochrome oxidase subunit 2 (Cox2) and cytochrome b2 (Cytb2). The molecular defect in an imp1 mutation and the characterisation of a high-copy-number suppressor is described. A deletion of the suppressor region causes respiration deficiency. The DNA sequence revealed three very small overlapping ORFs. Constructs which carried termination codons within the ORFs or lacked ATG initiation codons still retained complementing activity on a high-copy-number plasmid. Nevertheless, the possibility that the suppressor acts at DNA or RNA level could be excluded. Subcloning of the ORFs, complementation analysis in low-copy-number plasmids and transcript mapping identified the 222 bp ORF as the suppressor gene designated SOM1. The SOM1 gene is transcribed into a 375 bp polyadenylated RNA and the deduced amino acid sequence predicts a small protein of 8.4 kDa with no significant sequence similarity to known proteins. In the som1 deletion mutant, proteolytic processing of the Cox2 precursor is prevented and Cytb2 is strongly reduced. SOM1 represents a new small gene which encodes a novel factor that is essential for the correct function of the Imp1 peptidase and/or the protein sorting machinery.

Amino Acid Sequence↗
PubMed1986

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins.

Temperature-sensitive mutations in the RNA2 through RNA11 genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.

Cell Nucleus↗
PubMed2003

Unisexuality and molecular drive: bag320 sequence diversity in bacillus taxa (insecta phasmatodea).

Satellite DNA variability follows a pattern of concerted evolution through homogenization of new variants by genomic turnover mechanisms and variant fixation by chromosome redistribution into new combinations with the sexual process. Bacillus taxa share the same Bag320 satellite family and their reproduction ranges from strict bisexuality (B. grandii) to automictic (B. atticus) and apomictic (B. whitei = rossius/ grandii; B. lynceorum = rossius/grandii/atticus) unisexuality. Thelytokous reproduction clearly allows uncoupling of homogenization from fixation. Both trends and absolute values of satellite variability were analyzed in all Bacillus taxa but B. rossius, on 906 sequenced monomers at all level of comparisons: intraspecimen, intrapopulation, interpopulation, intersubspecies, and interspecies. For unisexuals, allozymic and mitochondrial clones were also taken into account. Different reproductive modes (sexual/parthenogenetic) appear to explain observed variability trends, supporting Dover's hypothesis of sexuality acting as a driving force in the fixation of sequence variants, but the present analyses also highlight current spreading of new variants in B. grandii maretimi specimens and point to a biased sequence inheritance at the time of hybrid onset in the apomictic hybrids B. whitei and B. lynceorum. Evidence of biased gene conversion events suggests that, given enough time, sequence homogenization can take place in a unisexual such as B. lynceorum. On the contrary, the absolute values of sequence diversity in each taxon are linked to the species' range, time of divergence, and repeat copy number and, possibly, to transposon features. Satellite dynamics appears therefore to be the outcome of both general molecular processes and specific organismal traits.

Animals↗

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