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PubMed2006

A maternal-offspring coadaptation theory for the evolution of genomic imprinting.

Imprinted genes are expressed either from the maternally or paternally inherited copy only, and they play a key role in regulating complex biological processes, including offspring development and mother-offspring interactions. There are several competing theories attempting to explain the evolutionary origin of this monoallelic pattern of gene expression, but a prevailing view has emerged that holds that genomic imprinting is a consequence of conflict between maternal and paternal gene copies over maternal investment. However, many imprinting patterns and the apparent overabundance of maternally expressed genes remain unexplained and may be incompatible with current theory. Here we demonstrate that sole expression of maternal gene copies is favored by natural selection because it increases the adaptive integration of offspring and maternal genomes, leading to higher offspring fitness. This novel coadaptation theory for the evolution of genomic imprinting is consistent with results of recent studies on epigenetic effects, and it provides a testable hypothesis for the origin of previously unexplained major imprinting patterns across different taxa. In conjunction with existing hypotheses, our results suggest that imprinting may have evolved due to different selective pressures at different loci.

Adaptation, Biological↗
PubMed2001

Signal processing by vestibular nuclei neurons is dependent on the current behavioral goal.

The vestibular sensory apparatus and associated vestibular nuclei are generally thought to encode angular head velocity during our daily activities. However, in addition to direct inputs from vestibular afferents, the vestibular nuclei receive substantial projections from cortical, cerebellar, and other brainstem structures. Given this diversity of inputs, the question arises: How are the responses of vestibular nuclei neurons to head velocity modified by these additional inputs during naturally occurring behaviors? Here we have focused on the signal processing done by two specific classes of neurons in the vestibular nuclei: (1) position-vestibular-pause (PVP) neurons that mediate the vestibulo-ocular reflex (VOR), and (2) vestibular-only (VO) neurons that are thought to mediate, at least in part, the vestibulo-collic reflex (VCR). We first characterized neuronal responses to passive rotation in the head-restrained condition, and then released the head to record the discharges of the same neurons during self-generated head movements. VOR interneurons (i.e., PVP neurons) faithfully transmitted head velocity signals when the animal stabilized its gaze, regardless of whether the head motion was actively or passively generated; their responses were attenuated only when the monkey's behavioral goal was to redirect its axis of gaze relative to space. In contrast, VCR interneurons (i.e., VO neurons) faithfully transmitted head velocity signals during passive head motion, but their responses were greatly (and similarly) attenuated during all behaviors (i.e., gaze shifts, gaze pursuit, gaze stabilization) during which the monkey's behavioral goal was to move its head relative to the body. To characterize the mechanism(s) that underlie this differential processing, we tested neurons during passive rotation of the head relative to the body, as well as during a task in which a monkey actively "drove" both its head and body together in space. We conclude that neither passive activation of neck proprioceptors nor knowledge of self-generated head-in-space motion directly mediate the observed reductions in head-velocity-related modulation. Instead, we propose that the VOR and VCR pathways use efference copies of oculomotor and neck movement commands, respectively, for the differential processing of vestibular information.

Action Potentials↗
PubMed1990

[A new laser scan system for video ophthalmoscopy. Initial clinical experiences also in relation to digital image processing].

The clinical advantages of a scanning laser ophthalmoscope (SLO) and video imaging of fundus pictures are described. Image quality (contrast, depth of field) and imaging possibilities (confocal stop) are assessed. Imaging with different lasers (argon, He-Ne) and changes in imaging rendered possible by confocal alignment of the imaging optics are discussed. Hard copies from video images are still of inferior quality compared to fundus photographs. Methods of direct processing and retrieval of digitally stored SLO video fundus images are illustrated by examples. Modifications for a definitive laser scanning system - in regard to the field of view and the quality of hard copies - are proposed.

Computer Systems↗
PubMed1982

Transposon-specified site-specific recombination.

Cointegrate DNA molecules containing two copies of a transposable element appear to be intermediates in the transposition process. These structures are resolved by site-specific recombination to yield the normal end products of transposition. The transposable element gamma delta (Tn1000) synthesizes a product interchangeable with the Tn1/3tnpR protein in promoting Tn1/3 site-specific recombination. These data support the hypothesis that cointegrates containing directly repeated copies of Tn1/3 are obligatory intermediates in interreplicon transposition of Tn1/3. In addition, we show here that the reaction is independent of the element-encoded tnpA gene product. Tn501, which specifies mercury resistance, also produces cointegrates as intermediates in interreplicon transposition. The appearance of Tn501-specified recombination activity that can act on these cointegrates requires growth of cells in the presence of Hg2+.

DNA Transposable Elements↗
PubMed2002

Determining the predictors of internal medicine residency accreditation: what they do (not what they say).

PURPOSE: The Accreditation Council for Graduate Medical Education and the Residency Review Committee for Internal Medicine (RRC-IM) evaluate internal medicine residency programs using a list of 301 program requirements. The authors investigated which requirements, program demographics, and site-visitor characteristics were the strongest predictors of accreditation. METHOD: The authors surveyed the program directors of all 405 accredited internal medicine residency programs in February 1998, obtaining data on the duration of the accreditation process, site visitors, and number and quality of citations. They also requested a copy of the notification letter containing citations and length of time until the next accreditation site visit (cycle length). RESULTS: A total of 217 responses (54%) was received. The mean cycle length was 3.0 years, and the accreditation process averaged 14.5 months. Smaller programs had a shorter average cycle length. Site visitors were reported to be prepared and professional overall. However, site visitors with the lowest evaluations by program directors were associated with shorter cycle lengths. Four program characteristics and program citations accounted for 60% of the variation in cycle length: total number of citations in the notification letter, percentage of graduates passing the American Board of Internal Medicine Certifying Examination, inadequate demonstration of resident scholarship, and inadequate ambulatory care experience. CONCLUSION: The authors devised an independent mechanism for determining the duration of the RRC-IM review process, influence of program demographics on the process, influence of site visitors on the accreditation action, and program requirements having the greatest effect on cycle length.

Accreditation↗
PubMed1997

Novel insulinoma cell lines produced by iterative engineering of GLUT2, glucokinase, and human insulin expression.

Cellular engineering studies in our group are directed at creating insulin-secreting cell lines that simulate the performance of the normal islet beta-cell. The strategy described in this article involves the stepwise stable introduction of genes relevant to beta-cell performance into the RIN 1046-38 insulinoma cell line, a process that we term "iterative engineering." RIN cells stably engineered to contain multiple copies of the human insulin gene exhibit a large increase in insulin content, such that they approach the content of human islets assayed in parallel. Analysis by high-performance liquid chromatography demonstrates that these engineered cell lines process human proinsulin to mature insulin with high efficiency. Cell lines that are further engineered to express the GLUT2 and glucokinase genes demonstrate stable expression of the three transgenes for the full lifetime of the lines produced to date (6 months to 1 year in continuous culture). Transplantation of the engineered cell lines into nude rats reveals that stably integrated genes are expressed at constant levels in the in vivo environment over the full duration of experiments performed (48 days). Several endogenous genes expressed in normal beta-cells, including rat insulin, amylin, sulfonylurea receptor, and glucokinase, are stably expressed in the insulinoma lines during these in vivo studies. Endogenous GLUT2 expression, in contrast, is rapidly extinguished during in vivo passage. The loss of GLUT2 is overcome in engineered cell ines in which transporter expression is provided by a stably transfected transgene. These results suggest that a potential advantage of the iterative engineering approach may be to preserve stability of function and phenotype, particularly in the in vivo setting.

Animals↗
PubMed1996

List update processing (LUP)--solving the sequence database update problem.

Sequence databases of today require frequent updating. Mirror procedures to copy incrementally updated databases as cumulative sets are the preferred method and can be implemented by straightforward scripting. However, limited bandwidth of networks and the increase of data require more powerful paradigms to reduce the workload reliably. We suggest the List Update Processing (LUP) principle. The system has been implemented on an experimental basis to update the Swiss EMBnet Node (BioComputing Basel, CH) with data from the European Bioinformatics Institute (EMBL Outstation, Hinxton Hall, UK). The results obtained from the prototype suggest to expand the system to several sites.

CD-ROM↗
PubMed1996

Identification of a differentiation-inducible promoter in the E7 open reading frame of human papillomavirus type 16 (HPV-16) in raft cultures of a new cell line containing high copy numbers of episomal HPV-16 DNA.

Gene expression of human papillomaviruses (HPV) is tightly linked to differentiation processes within the pluristratified epithelium. To analyze changes in the transcription pattern of HPV-16 during epithelial cell differentiation, we established a permanently growing HPV-16 positive cell line, designated KG, from a vulvar intraepithelial neoplasm. KG cells of early passages harbored multiple copies of the HPV-16 DNA as episomes and were able to form a stratified epithelium in an organotypic raft culture system. Analysis of viral gene expression revealed the known transcription pattern of the early region of HPV-16 with the exception of a so far undefined mRNA class with start sites in the E7 open reading frame. Quantitative analysis of primer extension experiments with RNA from KG cells grown in monolayer and raft culture showed a strong induction of this transcript in differentiated KG cells, whereas the level of the mRNAs initiated at the early promoter P97 remained almost constant. Primer extension analyses with four different primers and direct sequencing of the extension product revealed that the differentiation-inducible transcript initiated at a novel promoter with a major start site around nucleotide position 670 (P670) in the E7 open reading frame of HPV-16. Sequence analysis of cDNAs derived from RNA of KG cells grown in raft culture suggested that the transcripts initiated at P670 have a coding potential for an E1E4 fusion protein and for the E5 protein.

3T3 Cells↗
PubMed2004

Arf1p provides an unexpected link between COPI vesicles and mRNA in Saccharomyces cerevisiae.

The small GTPase Arf1p is involved in different cellular processes that require its accumulation at specific cellular locations. The recruitment of Arf1p to distinct points of action might be achieved by association of Arf1p with different proteins. To identify new interactors of Arf1p, we performed an affinity chromatography with GTP- or GDP-bound Arf1p proteins. A new interactor of Arf1p-GTP was identified as Pab1p, which binds to the polyA-tail of mRNAs. Pab1p was found to associate with purified COPI-coated vesicles generated from Golgi membranes in vitro. The stability of the Pab1p-Arf1p complex depends on the presence of mRNA. Both symmetrically distributed mRNAs as well as the asymmetrically localized ASH1 mRNA are found in association with Arf1p. Remarkably, Arf1p and Pab1p are both required to restrict ASH1 mRNA to the bud tip. Arf1p and coatomer play an unexpected role in localizing mRNA independent and downstream of the SHE machinery. Hereby acts the SHE machinery in long-range mRNA transport, whereas COPI vesicles could act as short-range and localization vehicles. The endoplasmic reticulum (ER)-Golgi shuttle might be involved in concentrating mRNA at the ER.

ADP-Ribosylation Factor 1↗
PubMed1991

Over-expression, purification and determination of the proteolytic processing site of the yeast mitochondrial CBS1 protein.

Yeast transformants harboring the CBS1 gene under the control of the strong ADC1 promoter on a high copy number plasmid express the mitochondrial CBS1 protein at artificially high levels. Over-expressed protein is imported into mitochondria and correctly processed to yield the mature mitochondrial 23.5 kDa form, but differs in its solubility properties from CBS1 in wild-type mitochondria. It forms insoluble protein aggregates, which are refractory to solubilization with 1% Taurodeoxycholate. We exploited this observation to separate CBS1 from the bulk of mitochondrial proteins and to isolate CBS1 after SDS gel electrophoresis. Determination of the amino-terminal amino acids of the purified protein reveals that the mature CBS1 protein starts with Ile30, at the characteristic distance of +2 amino acids from an arginine residue (Arg28). The cleavage site shows a remarkable homology to that of subunit 9 of the F0F1 ATPase from Neurospora crassa.

Amino Acid Sequence↗
PubMed1996

A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding.

Formation of non-clathrin-coated vesicles requires the recruitment of several cytosolic factors to the Golgi membrane. To identify membrane proteins involved in this budding process, a highly abundant type I transmembrane protein (p23) was isolated from mammalian Golgi-derived COPI-coated vesicles, and its cDNA was cloned and sequenced. It belongs to the p24 family of proteins involved in the budding of transport vesicles (Stamnes, M.A., M.W. Craighead, M.H. Hoe, N. Lampen, S. Geromanos, P. Tempst, and J.E. Rothman. 1995. Proc. Natl. Acad. Sci. USA. 92:8011-8015). p23 consists of a large NH2-terminal luminal domain and a short COOH-terminal cytoplasmic tail (-LRRFFKAKKLIE-CO2-) that shows similarity, but not identity, with the sequence motif-KKXX-CO2-, known as a signal for retrieval of escaped ER-resident membrane proteins (Jackson, M.R., T. Nilsson, and P.A. Peterson. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:3153-3162; Nilsson, T., M. Jackson, and P.A. Peterson. 1989. Cell. 58:707-718). The cytoplasmic tail of p23 binds to coatomer with similar efficiency as known KKXX motifs. However, the p23 tail differs from the KKXX motif in having an additional motif needed for binding of coatomer. p23 is localized to Golgi cisternae and, during vesicle formation, it concentrates into COPI-coated buds and vesicles. Biochemical analysis revealed that p23 is enriched in vesicles by a factor of approximately 20, as compared with the donor Golgi fraction, and is present in amounts stoichiometric to the small GTP-binding protein ADP-ribosylation factor (ARF) and coatomer. From these data we conclude that p23 represents a Golgi-specific receptor for coatomer involved in the formation of COPI-coated vesicles.

Amino Acid Sequence↗
PubMed1995

Optimization of glucose oxidase production by Aspergillus niger using genetic- and process-engineering techniques.

Wild-type Aspergillus niger NRRL-3 was transformed with multiple copies of the glucose oxidase structural gene (god). The gene was placed under the control of the gpdA promoter of A. nidulans. For more efficient secretion the alpha-amylase signal peptide from A. oryzae was inserted in front of god. Compared to the wild type, the recombinant strain NRRL-3 (GOD3-18) produced up to four times more extracellular glucose oxidase under identical culture conditions. Addition of yeast extract (2 gl-1) to a mineral salts medium containing only glucose as carbon source increased volumetric and specific extracellular glucose oxidase activities by 130% and 50% respectively. With the same medium composition and inoculum size, volumetric and specific extracellular glucose oxidase activities increased more than ten times in bioreactor cultivations compared to shake-flask cultures.

Aspergillus nidulans↗
PubMed1993

A regulatory function for K10 in the establishment of dorsoventral polarity in the Drosophila egg and embryo.

Several lines of evidence suggest that the origin of pattern formation of Drosophila embryos must be traced back to oogenesis, to the polarity of the egg chamber. A few early-acting genes, K10, top, grk and cni, have been identified which are assumed to function in a signal transduction process between the germline oocyte and the somatic follicle cells, during which the egg chamber acquires a dorsoventral polarity. K10 has been cloned and was shown to encode a putative transcription factor specifically acting in the oocyte nucleus. In order to characterize further the function of K10, we have analyzed its genetic interactions with grk, top and cni. We show that grk behaves as a dominant partial suppressor of K10. Analysis of the rescuing process of the K10 phenotype by grk shows that: (1) K10 is not indispensable for the establishment of dorsoventral polarity of the egg chamber, since its lack of function can be compensated for by reducing the grk wild-type copy number; (2) grk function is highly dose-sensitive; (3) the rescue process shows an anteroposterior effect suggesting that K10 may also interact with genes involved in anteroposterior pattern formation. These results are compatible with a model in which grk is a dorsalizing signal emanating from the oocyte nucleus, whose level of expression is regulated negatively by the K10 product.

Animals↗
PubMed1995

Quality control of storage phosphor digital radiography systems.

Quality control (QC) of storage phosphor devices is important in assuring that the image information entered into an Image management and communication (IMAC) system is sufficient for diagnosis. QC of storage phosphor digital radiography systems is complex because of the self-corrective nature of the image-processing software used in these machines. Currently, one must produce hard copy to perform adequate QC. Inspection of images with reject analysis and inspection of cassettes and imaging plates has helped us in our QC program. For those QC tests using control limits, the appropriate settings for these limits are unknown. Starting approximations are given. Recommended tests are described.

Equipment Failure↗
PubMed1991

5' flanking sequences of human MRP/7-2 RNA gene are required and sufficient for the transcription by RNA polymerase III.

Human mitochondrial RNA processing (MRP) RNA is a 270 nucleotide-long small RNA found as ribonucleoprotein particles. In this study, we isolated four human genomic clones with homology to human MRP RNA. Two of these clones contained one copy each of the real gene coding for human MRP RNA; the other two clones represented a processed psuedogene. The Southern blot with the genomic DNA showed that the haploid human genome contains one copy of real gene and a few pseudogenes for MRP/7-2 RNA. The human MRP RNA is synthesized by RNA polymerase III and the 5' flanking sequences -84 to 1 of MRP RNA gene, containing TATA and PSE-like elements, are required and sufficient for transcription in vitro.

Animals↗
PubMed1992

A spontaneous chromosomal amplification of the ADH2 gene in Saccharomyces cerevisiae.

A spontaneous antimycin A-resistant mutant carrying approximately four extra copies of ADH2 on chromosome XII was isolated from yeast strain 315-1D which lacks a functional copy of ADH1 and thus is antimycin A-sensitive. The additional copies of the normally glucose-repressed ADH2 are expressed during growth on glucose accounting for the antimycin A resistance. These extra copies are inserted into nonadjacent ribosomal DNA sequences (rDNA) near the recombination stimulating sequence HOT1. Each extra copy of the ADH2 gene (1548 bp) replaces most of the 37S transcript (approximately 7400 bp) in one of the approximately 200 copies of the rDNA present in the yeast genome. All four extra copies of ADH2 are lost at a rate of approximately 1 x 10(-5) deletions per cell per generation. One of the joints between the rDNA and ADH2 DNA is located 7 nucleotides downstream from 20 adenine residues in the normal copy of ADH2. This joint occurs at the end of a stretch of 16-29 thymidines in the rDNA which has been expanded to 57-59 thymidines. The other novel joint is located in a short region of sequence similarity between ADH2 and the rDNA. These observations suggest that amplification of ADH2 was a two step process: first the ADH2 gene was inserted into the rDNA, then multiple copies were generated by unequal crossing over or gene conversion within the rDNA.

Alcohol Dehydrogenase↗
PubMedDate not supplied

Epigenetic regulation of programmed genomic rearrangements in Paramecium aurelia.

In ciliates, development of the polyploid somatic macronucleus after sexual events involves extensive and reproducible rearrangements of the germ-line genome, including chromosome fragmentation and precise excision of numerous internal sequence elements. In Paramecium aurelia, alternative macronuclear versions of the same germ-line genome can be maternally inherited across sexual generations, showing that rearrangement patterns are not strictly determined by the germ-line sequence. Homology-dependent maternal effects can be evidenced by transformation of the vegetative macronucleus with cloned macronuclear sequences: new fragmentation patterns or internal deletions are specifically induced during differentiation of a new macronucleus, in sexual progeny of transformed clones. Furthermore, transformation of the maternal macronucleus with germ-line sequences containing internal eliminated sequences (short single-copy elements) can result in a specific inhibition of the excision of the same elements in the zygotic macronucleus. These experiments show that the processing of many germ-line sequences in the developing macronucleus is sensitive to the structure and copy number of homologous sequences in the maternal macronucleus. The generality and sequence specificity of this trans-nuclear, epigenetic regulation of rearrangements suggest that it is mediated by pairing interactions between germ-line sequences and sequences imported from the maternal macronucleus.

Animals↗
PubMed1996

Human papilloma virus detection by in situ hybridisation signal amplification based on biotinylated tyramine deposition.

Aim-To describe a method for amplifying human papilloma virus (HPV) in situ hybridisation (ISH) signals.Methods-Three human cervical cell lines, namely CaSKi, HeLa and SiHa, containing different copy numbers of integrated HPV DNA were studied. Following ISH, catalysed reporter deposition (CARD), based on the deposition of biotinylated tyramine at the location of the DNA probe, was used to amplify the ISH signal.Results-Using CARD-ISH, one to three HPV type 16 copies were detected in situ both in cell suspensions and paraffin wax sections of SiHa cells. CARD-ISH can also be used to detect oncogenic HPV DNA sequences, such as HPV types 16 and 18, in routinely processed formalin fixed, paraffin wax embedded cervical specimens.Conclusions-CARD-ISH is a fast and highly sensitive ISH method for the routine detection of low copy number HPV DNA sequences in cervical cell lines and routinely processed tissue sections. Application of this technology also enables the routine detection and cellular localisation of other viral DNA sequences present at copy numbers below the detection limit of conventional ISH methods.

Journal Article↗