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At least 1,333 records · Page 74Linked to original sources
PubMed2005

Mass spectrometrical analysis of recombinant human growth hormone (Genotropin(R)) reveals amino acid substitutions in 2% of the expressed protein.

BACKGROUND: The structural integrity of recombinant proteins is of critical importance to their application as clinical treatments. Recombinant growth hormone preparations have been examined by several methodologies. In this study recombinant human growth hormone (rhGH; Genotropin(R)), expressed in E. coli K12, was structurally analyzed by two-dimensional gel electrophoresis and MALDI-TOF-TOF, LC-MS and LC-MS/ MS sequencing of the resolved peptides. RESULTS: Electrospray LC-MS analysis revealed one major protein with an average molecular mass of 22126.8 Da and some additional minor components. Electrospray LC-MS/MS evaluation of the enzymatically digested Genotropin(R) sample resulted in the identification of amino acid substitutions at the residues M14, M125, and M170; di-methylation of K70 (or exchange to arginine); deamidation of N149, and N152, and oxidation of M140, M125 and M170. Peak area comparison of the modified and parental peptides indicates that these changes were present in ~2% of the recombinant preparation. CONCLUSION: Modifications of the recombinant human growth hormone may lead to structural or conformational changes, modification of antigenicity and development of antibody formation in treated subjects. Amino acid exchanges may be caused by differences between human and E. coli codon usage and/or unknown copy editing mechanisms. While deamidation and oxidation can be assigned to processing events, the mechanism for possible di-methylation of K70 remains unclear.

Journal Article↗
PubMed2005

A mutation in the centriole-associated protein centrin causes genomic instability via increased chromosome loss in Chlamydomonas reinhardtii.

BACKGROUND: The role of centrioles in mitotic spindle function remains unclear. One approach to investigate mitotic centriole function is to ask whether mutation of centriole-associated proteins can cause genomic instability. RESULTS: We addressed the role of the centriole-associated EF-hand protein centrin in genomic stability using a Chlamydomonas reinhardtii centrin mutant that forms acentriolar bipolar spindles and lacks the centrin-based rhizoplast structures that join centrioles to the nucleus. Using a genetic assay for loss of heterozygosity, we found that this centrin mutant showed increased genomic instability compared to wild-type cells, and we determined that the increase in genomic instability was due to a 100-fold increase in chromosome loss rates compared to wild type. Live cell imaging reveals an increased rate in cell death during G1 in haploid cells that is consistent with an elevated rate of chromosome loss, and analysis of cell death versus centriole copy number argues against a role for multipolar spindles in this process. CONCLUSION: The increased chromosome loss rates observed in a centrin mutant that forms acentriolar spindles suggests a role for centrin protein, and possibly centrioles, in mitotic fidelity.

Animals↗
PubMed2006

Executive functions in becoming writing readers and reading writers: note taking and report writing in third and fifth graders.

Results are reported for a study of 2 separate processes of report writing-taking notes while reading source material and composing a report from those notes-and related individual differences in executive functions involved in integrating reading and writing during these writing activities. Third graders (n = 122) and 5th graders (n = 106; overall, 127 girls and 114 boys) completed two reading-writing tasks-read paragraph (mock science text)-write notes and use notes to generate written report, a reading comprehension test, a written expression test, four tests of executive functions (inhibition, verbal fluency, planning, switching attention), and a working memory test. For the read-take notes task, the same combination of variables was best (explained the most variance and each variable added unique variance) for 3rd graders and 5th graders: Wechsler Individual Achievement Test-Second Edition (WIAT-II) Reading Comprehension, Process Assessment of the Learner Test for Reading and Writing (PAL) Copy Task B, WIAT-II Written Expression, and Delis-Kaplan Executive Function System (D-KEFS) Inhibition. For the use notes to write report task, the best combinations of variables depended on grade level: For 3rd graders, WIAT-II Reading Comprehension, WIAT-II Written Expression, D-KEFS Verbal Fluency, and Tower of Hanoi; for 5th graders, WIAT-II Reading Comprehension, D-KEFS Verbal Fluency, WIAT-II Written Expression, and PAL Alphabet Task. These results add to prior research findings that executive functions contribute to the writing development of elementary-grade students and additionally support the hypothesis that executive functions play a role in developing reading-writing connections.

Analysis of Variance↗
PubMed1976

Molecular biology and biochemistry of differentiation.

Recent work in the field of molecular biology and differentiation has been directed towards an assessment of the number of different genes involved in the development and differentiation process. By the techniques of RNA-DNA hybridization to single copy DNA, it appears that some 60,000-200,000 different RNA sequences are expressed during embryonic development in the mouse. The differentiating brain shows the highest degree RNA transcription diversity. The new technique of cDNA-poly(A)-containing RNA hybridization is described. The nuclear poly(A)-containing RNA appears to reflect the high complexity of sequences as determined in RNA-DNA experiments described above. The cytoplasmic poly(A)-containing RNA or messenger RNA appears to represent a subset of approximately 10-20% of the information in the nuclear poly (A)-RNA. Also, the major portion of frequency class of messenger RNA in different organs such as kidney, spleen, and liver are common to these different organs. However, brain, although containing many of the cytoplasmic messenger RNA sequences found in liver, kidney, and spleen, has a large class of messenger RNA sequences which are specific to the nervous system.

Animals↗
PubMed2003

Analysis of large structural changes of the factor VIII gene, involving intron 1 and 22, in severe hemophilia A.

BACKGROUND AND OBJECTIVES: Hemophilia A (HA), the deficiency of coagulation factor VIII (FVIII), is the most common, sex-linked inherited bleeding disorder. The disease is caused by FVIII gene intron 22 inversion in approximately 50% of the patients, and by intron 1 inversion in 5% of the patients with severe HA. Both inversions occur as a result of intrachromosomal recombination between homologous regions, in intron 1 or 22, and their extragenic copy located telomeric to the FVIII gene. The goal of the present study was to analyze the presence of large structural changes in the FVIII gene in patients with severe hemophilia A. DESIGN AND METHODS: We studied 104 unrelated, severe HA-patients or obligate carriers for the presence of intron 22 and intron 1 inversions by Southern blotting, long-distance polymerase chain reaction (PCR), and simple PCR. RESULTS: We found altered intron 22 restriction profiles by Southern analyses in 58 cases: 43 type 1, 11 type 2 inversions and 4 unusual patterns. Upon further examination of the last 4 cases, large deletions involving intron 22 were demonstrated in two cases. In the remaining two patients extra homologous regions were detected by Southern analysis, and long-distance PCR showed the presence of unaltered intra- and extragenic copies together with one inversion-affected copy, suggesting that an additional intronic fragment participated in the inversion process and was inserted in the genome. During screening for intron 1 inversion among 43 patients, who were intron 22 inversion negative, we identified only wild type individuals. INTERPRETATION AND CONCLUSIONS: The relatively large proportion of unusual patterns further supports the observation that the structure of FVIII intron 22 represents a hot spot for large gene rearrangements with various mechanisms, while intron 1 inversion seems to be not common in Hungary.

Adolescent↗
PubMed1992

Characterization of a cDNA clone encoding multiple copies of the neuropeptide APGWamide in the mollusk Lymnaea stagnalis.

Male mating behavior of the simultaneous hermaphrodite freshwater snail Lymnaea stagnalis is controlled by a neuronal network that consists of various types of peptidergic neurons, as well as serotonergic cells. In the present article, we describe the isolation and characterization of a cDNA clone that encodes a multipeptide preprohormone expressed in the anterior lobe of the right cerebral ganglion, in a group of neurons that principally innervate the penial complex. The preprohormone is 219 amino acids in length and contains 10 copies of the peptide Ala-Pro-Gly-Trp-Gly. Posttranslational processing of the prohormone may lead to the generation of the amidated neuropeptide Ala-Pro-Gly-Trp-amide (APGWamide), an amidated C-terminal anterior lobe peptide, and four connecting peptide sequences, C1-C4. We show by in situ and filter hybridizations that neurons of the right anterior lobe comprise the major site of expression of the APGWamide gene. Expression of the APGWamide gene is detected in the CNS of both adult animals and noncopulating juveniles. Peptides derived from the APGWamide prohormone are probably involved in the control of a part of the male mating behavior and have both central and peripheral targets.

Amino Acid Sequence↗
PubMed1983

Use of a solid-state multihead gamma counter in a second-generation system for solid-phase immunoassay.

Simultaneous advances in detector technology and solid-phase separation systems, as well as the availability of powerful desktop computers, have made possible the development of "second-generation" solid-phase immunoassays. These retain the advantages of classical solid phase while significantly accelerating reaction kinetics. Hapten assays--such as for digoxin, thyroxin, and triiodothyronine uptake--in batches of 48 are processed in about 20 min from reagent introduction until hard-copy printout, with minimal operator involvement. The system also functions as a 48-detector gamma counter, capable of counting and reducing data for any 125I-based RIA that can be run in a 12 X 75 mm test tube. System control, data management, and computer screen displays of kinetic data are provided by an unmodified Hewlett Packard HP-87XM computer. User-friendly disc-based software facilitates the creation and storage of counting and data reduction protocols for as many as 30 RIAs from various manufacturers as well as up to 30 of our own assays.

Computers↗
PubMed2004

Modeling senescence in hypotrichous ciliates.

A sexually reproducing hypotrichous ciliates undergo senescence which is in general attributed to degenerative processes in the macronucleus, assuming that loss of viability is based on loss of genetic elements. It is generally accepted that the genetic elements in the macronucleus of hypotrichs segregate randomly, a process which potentially can lead to aneuploid imbalances in the distribution of gene copies. It is, however, unclear whether there are mechanisms which compensate for such imbalances such that each genetic element regains its predetermined copy number (regulatory model, conserving euploidy), or whether the genetic elements only double, so that genetic imbalances can be inherited to further generations (stochastic model, allowing aneuploidy). By means of mathematical modeling and simulations, we investigate these two models with respect to the number of generations a lineage of hypotrichs can survive under asexual conditions. Whereas the regulatory model cannot explain senescence in hypotrichs, the stochastic model provides plausible results which, however, strongly depend on the assumed distribution of copy numbers which we investigate by means of three examples. For both models, simple prediction formulae for the approximate survival time of asexually reproducing ciliates are provided.

Aging↗
PubMed1998

Role of the region 3' to Xist exon 6 in the counting process of X-chromosome inactivation.

During early embryogenesis of female mammals, one of the two X chromosomes is randomly chosen to be inactivated in each cell, leading to the transcriptional silencing of thousands of genes on this chromosome. This random X-inactivation process also occurs during in vitro differentiation of female embryonic stem (ES) cells. A locus on the X chromosome, the X inactivation centre (Xic) is initially 'counted', given that at least two copies of Xic must be present per diploid genome in order for inactivation to occur. The counting process ensures that one X chromosome remains active in diploid cells. In the mouse, the essential functions of Xic can be assured by a 450-kb region containing the Xist gene. Xist maps within Xic (refs 7-10) and is necessary in cis for inactivation. The Xist transcript is a 15-kb RNA which is confined within the nucleus and coats the inactive X chromosome. In order to characterize functional elements within Xic and the Xist gene, we created a 65-kb cre/loxP deletion extending 3' to Xist exon 6. In undifferentiated ES cells, Xist expression from the deleted X chromosome was markedly reduced. In differentiated XX ES cells containing one deleted X chromosome, the X inactivation process still occurred but was never initiated from the unmutated X chromosome. In differentiated ES cells that were essentially XO, the mutated Xic was capable of initiating X inactivation, even in the absence of another Xic. These results demonstrate a role for the region 3' to Xist exon 6 in the counting process and suggest that counting is mediated by a repressive mechanism which prevents inactivation of a single X chromosome in diploid cells.

A Kinase Anchor Proteins↗
PubMed2025

Single-cell copy number calling and event history reconstruction.

MOTIVATION: Copy number alterations are driving forces of tumour development and the emergence of intra-tumour heterogeneity. A comprehensive picture of these genomic aberrations is therefore essential for the development of personalised and precise cancer diagnostics and therapies. Single-cell sequencing offers the highest resolution for copy number profiling down to the level of individual cells. Recent high-throughput protocols allow for the processing of hundreds of cells through shallow whole-genome DNA sequencing. The resulting low read-depth data poses substantial statistical and computational challenges to the identification of copy number alterations. RESULTS: We developed SCICoNE, a statistical model and MCMC algorithm tailored to single-cell copy number profiling from shallow whole-genome DNA sequencing data. SCICoNE reconstructs the history of copy number events in the tumour and uses these evolutionary relationships to identify the copy number profiles of the individual cells. We show the accuracy of this approach in evaluations on simulated data and demonstrate its practicability in applications to two breast cancer samples from different sequencing protocols. AVAILABILITY AND IMPLEMENTATION: SCICoNE is available at https://github.com/cbg-ethz/SCICoNE.

Single-Cell Analysis↗
PubMed1995

The contribution of slippage-like processes to genome evolution.

Simple sequences present in long (> 30 kb) sequences representative of the single-copy genome of five species (Homo sapiens, Caenorhabditis elegans, Saccharomyces cerevisiae, E. coli, and Mycobacterium leprae) have been analyzed. A close relationship was observed between genome size and the overall level of sequence repetition. This suggested that the incorporation of simple sequences had accompanied increases of genome size during evolution. Densities of simple sequence motifs were higher in noncoding regions than in coding regions in eukaryotes but not in eubacteria. All five genomes showed very biased frequency distributions of simple sequence motifs in all species, particularly in eukaryotes where AAA and TTT predominated. Interspecific comparisons showed that noncoding sequences in eukaryotes showed highly significantly similar frequency distributions of simple sequence motifs but this was not true of coding sequences. ANOVA of the frequency distributions of simple sequence motifs indicated strong contributions from motif base composition and repeat unit length, but much of the variation remained unexplained by these parameters. The sequence composition of simple sequences therefore appears to reflect both underlying sequence biases in slippage-like processes and the action of selection. Frequency distributions of simple sequence motifs in coding sequences correlated weakly or not at all with those in noncoding sequences. Selection on coding sequences to eliminate undesirable sequences may therefore have been strong, particularly in the human lineage.

Animals↗
PubMed1991

A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration.

A CHO cell line with a single copy of the DHFR locus on chromosome Z2 was used to analyze the structure of the amplification target and products subsequent to the initial amplification event. Dramatic diversity in the number and cytogenetic characteristics of DHFR amplicons was observed as soon as eight to nine cell doublings following the initial event. Two amplicon classes were noted at this early time: Small extrachromosomal elements and closely spaced chromosomal amplicons were detected in 30-40% of metaphases in six of nine clones, whereas three of nine clones contained huge amplicons spanning greater than 50 megabases. In contrast, the incidence of metaphases containing extrachromosomal amplicons fell to 1-2% in cells analyzed at 30-35 cell doublings, and most amplicons localized to rearranged or broken derivatives of chromosome Z2 at this time. Breakage of the Z2 chromosome near the DHFR gene, and deletion of the DHFR gene and flanking DNA was also observed in cells that had undergone the amplification process. To account for these diverse cytogenetic and molecular consequences of gene amplification, we propose that chromosome breakage plays a central role in the amplification process by (1) generating intermediates that are initially acentric and lead to copy number increase primarily by unequal segregation, (2) creating atelomeric ends that are either incompletely replicated or resected by exonucleases to generate deletions, and (3) producing recombinogenic ends that provide preferred sites for amplicon relocalization.

Animals↗
PubMed1998

Translational control of mRNA processing in the F1845 fimbrial operon of Escherichia coli.

Endoribonucleolytic processing followed by differential decay of the cleavage products is an increasingly recognized mechanism for achieving co-ordinate regulation of functionally related proteins encoded by bacterial polycistronic transcripts. Unlike most examples when RNases E or III initiate decay, the daa transcript encoding F1845 fimbriae, a member of the Dr family of adhesins in Escherichia coli, is processed by an as yet unidentified endoribonuclease using a unique recognition mechanism. An open reading frame (ORF) predicted to encode a 57-amino-acid polypeptide was identified flanking the daa processing site. To determine whether this ORF is involved in processing, site-directed mutagenesis was used to generate mutants with altered translational efficiencies. A mutation in the putative ribosome binding site preceding the ORF significantly inhibited processing while the introduction of a premature stop codon abolished processing. Site-directed mutagenesis was used to introduce a limited number of mutations into the ORF, designated daaP, to alter the reading frame such that a different polypeptide of a similar size was encoded. Despite the presumed presence of trafficking ribosomes, this mutant failed to be processed, suggesting that the sequence of the DaaP peptide is important. However, the failure of a wild-type copy of the daaP gene to complement these mutations in trans suggested that the presence of wild-type daaP gene product was not sufficient to promote processing. Although active translation has been found to inhibit processing by RNases E and III, our data suggest that translation of the daaP gene is required in cis to promote processing by the endonuclease, perhaps due to an interaction of the nascent peptide with the ribosome or the daaP mRNA.

Amino Acid Sequence↗
PubMedDate not supplied

PC12 cells can be induced to produce, but do not process, the neurotensin/neuromedin N precursor.

Neurotensin and neuromedin N are two biologically active, related peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-terminal region one copy each of neurotensin and neuromedin N. Four Lys-Arg sequences, which are thought to represent putative processing sites, occur in the precursor molecule. Studies by others have shown that rat pheochromocytoma PC12 cells produced neurotensin and dramatically increased their neurotensin/neuromedin N precursor mRNA content in response to a combination of nerve growth factor, dexamethasone, forskolin, and Li+. Here, we investigated the effects of this combination of inducers on the posttranslational processing of the neurotensin/neuromedin N precursor in PC12 cells. Radioimmunoassays coupled to HPLC and arginine-directed tryptic cleavage of cell extracts were performed with five antisera specific for precursor sequences adjacent to basic doublets. Thus, mature neurotensin and neuromedin N represented less than 1% of the total precursor content in PC12 cells. The PC12 cell line may represent an interesting model with which one could transfect the recently cloned prohormone convertases PC1 and PC2, thereby allowing the study of the role of these enzymes in the processing of the neurotensin/neuromedin N precursor.

Amino Acid Sequence↗
PubMed1987

Effects of recombinant plasmid size on cellular processes in Escherichia coli.

The effects of recombinant plasmid size on cell growth and viability, plasmid copy number, and synthesis of plasmid-encoded protein were investigated in Escherichia coli using plasmid pUC8 and four recombinant derivatives containing inserts of Drosophila melanogaster DNA of 1.7-6.0 kb. Growth in log phase was unaffected by plasmid size, but as plasmid size increased, maximum cell density decreased and, with the largest plasmid, cell death was accelerated after the stationary phase was reached. There was also a correlation between increasing plasmid size and decreased viability at high ampicillin concentrations, resistance to which is conferred by the plasmids. These effects were shown not to be due to transcription or translation of Drosophila sequences carried on the recombinant plasmids. Cells harboring the largest plasmid, pBS5 (8.7 kb), fared poorly in competition with plasmid-free cells in mixed cultures, compared with cells harboring pUC8 (2.7 kb). In addition, pBS5 was harbored at significantly fewer copies per cell than pUC8 at all phases of growth and supported much less production of the plasmid-encoded protein, beta-lactamase, than did pUC8. The results suggest that recombinant plasmid size may be an important parameter in the optimization of large-scale production of plasmid-encoded proteins.

DNA, Recombinant↗
PubMed2003

Mutations in the C-terminal domain of ALSV (Avian Leukemia and Sarcoma Viruses) integrase alter the concerted DNA integration process in vitro.

Integrase (IN) is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cell DNA. The C-terminal domain of IN is involved in DNA binding and enzyme multimerization. We previously performed single amino acid substitutions in the C-terminal domain of the avian leukemia and sarcoma viruses (ALSV) IN. Here, we modelled these IN mutants and analysed their ability to mediate concerted DNA integration (in an in vitro assay) as well as to form dimers (by size exclusion chromatography and protein-protein cross-linking). Mutations of residues located at the dimer interface (V239, L240, Y246, V257 and K266) have the greatest effects on the activity of the IN. Among them: (a) the L240A mutation resulted in a decrease of integration efficiency that was concomitant with a decrease of IN dimerization; (b) the V239A, V249A and K266A mutants preferentially mediated non-concerted DNA integration rather than concerted DNA integration although they were found as dimers. Other mutations (V260E and Y246W/DeltaC25) highlight the role of the C-terminal domain in the general folding of the enzyme and, hence, on its activity. This study points to the important role of residues at the IN C-terminal domain in the folding and dimerization of the enzyme as well as in the concerted DNA integration of viral DNA ends.

Alpharetrovirus↗
PubMed1992

Use of a cis-acting mutation to study the role of FLP-mediated recombination in the maintenance of native yeast 2 micrometer plasmids.

The 2 micrometer plasmid encodes a mechanism that ensures the partitioning of the plasmid at cell division. Little is known about the detailed mechanism of this partitioning system; for example, is there equal or unequal distribution of the plasmid molecules at mitosis? The plasmid also encodes a site-specific recombination system that is thought to be involved in plasmid copy-number amplification, although to date there has been no direct evidence that the recombination process itself is important for maintenance. We have identified a natural 2 micrometer variant that has a cis-acting mutation in the FLP-mediated recombination system. We show that this plasmid is unable to amplify in vivo. Our results demonstrate that the average copy number per cell is not affected for the mutant but there is a large clonal variation. This is a direct demonstration that plasmid partitioning results in an unequal distribution of plasmids and that FLP-mediated amplification compensates for this and therefore has an important role in maintenance.

DNA Nucleotidyltransferases↗
PubMed1997

The Rho-GEF Rom2p localizes to sites of polarized cell growth and participates in cytoskeletal functions in Saccharomyces cerevisiae.

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1 delta, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11 degrees C and 37 degrees C. rom2 delta cells exhibit morphological defects. At permissive temperatures, rom2 delta cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1 delta and kar3 delta strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore, rom2 delta strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton.

Binding Sites↗