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PubMed1988

Isolation and mapping of a gene for protein synthesis initiation factor 4A and its expression during differentiation of murine erythroleukemia cells.

Eukaryotic protein synthesis initiation factor 4A (eIF-4A), a 46-kDa polypeptide, is involved both in mRNA cap recognition and in the binding of mRNA to 40S ribosomal subunits. A 41-mer oligodeoxynucleotide probe was synthesized complementary to a portion of the published coding sequence of eIF-4A mRNA [Nielsen et al., Nucleic Acids Res. 13 (1985) 6867-6870] and used to screen a mouse genomic library. We have isolated and characterized a full-length clone from that library. The eIF-4A sequence is contained in eleven exons. The eleventh exon also has the 3'-nontranslated sequence and two separate polyadenylation sites. Northern-blot analysis of mouse poly(A)+RNA indicates that there are several distinct mRNA species coding for eIF-4A. Two of these contain the same coding sequence and differ only in the length of the 3'-nontranslated region. Two of the eIF-4A mRNAs are therefore likely to be the result of differential processing at the 3'-end. We have used a fragment of the genomic clone to measure the steady-state levels of eIF-4A mRNA during the induced differentiation of murine erythroleukemia cells. S1 nuclease protection experiments demonstrated that by the fourth day after induction eIF-4A mRNA declined to 25% of its steady-state level in uninduced cells. In contrast, the steady-state level of beta-globin mRNA increased dramatically during differentiation. In vitro transcription assays using nuclei isolated from uninduced and induced cells show that the rate of transcription of eIF-4A mRNA was 40% greater in differentiated cells, indicating a posttranscriptional component is involved in the regulation of the steady-state mRNA level.

Amino Acid Sequence↗
PubMed2005

Protein fragment complementation in M.HhaI DNA methyltransferase.

The 5mC DNA methyltransferase M.HhaI can be split into two individually inactive N- and C-terminal fragments that together can form an active enzyme in vivo capable of efficiently methylating DNA. This active fragment pair was identified by creating libraries of M.HhaI gene fragment pairs and then selecting for the pairs that code for an active 5mC methyltransferase. The site of bisection for successful protein fragment complementation in M.HhaI was in the variable region near the target recognition domain between motif VIII and TRD. This same region is the location of bifurcation in the naturally split 5mC methyltransferase M.AquI, the location for circular permutation in M.BssHII, and the location for previously engineered split versions of M.BspRI.

Amino Acid Sequence↗
PubMed2005

AutoLink: automated sequential resonance assignment of biopolymers from NMR data by relative-hypothesis-prioritization-based simulated logic.

We have developed a new computer algorithm for determining the backbone resonance assignments for biopolymers. The approach we have taken, relative hypothesis prioritization, is implemented as a Lua program interfaced to the recently developed computer-aided resonance assignment (CARA) program. Our program can work with virtually any spectrum type, and is especially good with NOESY data. The results of the program are displayed in an easy-to-read, color-coded, graphic representation, allowing users to assess the quality of the results in minutes. Here we report the application of the program to two RNA recognition motifs of Apobec-1 Complementation Factor. The assignment of these domains demonstrates AutoLink's ability to deliver accurate resonance assignments from very minimal data and with minimal user intervention.

Algorithms↗
PubMed2003

Rat and mouse membrane aminopeptidase P: structure analysis and tissue distribution.

Membrane-bound aminopeptidase P (mAPP) is a highly specific exopeptidase that removes the N-terminal amino acid only from a peptide (three amino acids or longer) that has a prolyl residue in the second position. mAPP can inactivate bradykinin, a potent vasodilating and cardioprotective peptide hormone, by hydrolyzing the Arg(1)-Pro(2) bond. Studies on the rat have shown that the metabolism of bradykinin is an important physiological role of this enzyme. We report here the complete coding sequences for rat and mouse mAPP determined from mRNA isolated from lung tissue. Key structural features that determine post-translational processing and substrate recognition and catalysis were identified based on sequence homologies and the crystal structure of Escherichia coli aminopeptidase P complexed with Pro-Leu. The tissue-specific expression of mAPP was studied using the polymerase chain reaction. The mAPP gene is widely, but variably, expressed in adult tissues of the rat and mouse and in mouse embryos.

Amino Acid Sequence↗
PubMed2001

The gene structure and minimal promoter of the human agouti related protein.

The murine agouti related protein (mAGRP) is upregulated in obese and diabetic mice and stimulates hyperphagia when administered intracerebroventricularly (i.c.v.) or when overexpressed in transgenic mice. The human ortholog, hAGRP, has been isolated and has similar molecular and physiological properties. Here, we report the complete gene structure of the human AGRP gene and upstream regions with differential promoter activity. A polymorphism, A67T, in the third exon was identified but was not associated with obesity- or type 2 diabetes-related phenotypes. Putative binding sites for transcription factors were identified in the promoter of the gene including recognition sites for the signal transducers and activators of transcription (STATs) that may potentially mediate leptin's action in the hypothalamus. The upstream non-coding exon had significant promoter activity in a periphery- but not so in a hypothalamus-derived cell line, suggesting that it might contain the minimal promoter required for expression of the short transcript of hAGRP in the periphery.

Agouti-Related Protein↗
PubMed2004

Reporting instruction for radiology residents.

RATIONALE AND OBJECTIVES: To determine the amount of formal instruction and evaluation about reporting given to radiology residents in the U.S.A., to document report generation methods and to quantify the performance of physician coding. MATERIALS AND METHODS: E-mail requests with links to a web-based, anonymous survey were sent to program directors of all accredited radiology residencies in the USA. Demographic questions included university or private affiliation, number of residents, geographic location, and number of hospitals covered. Subject-specific items covered the amount of didactic instruction, formal evaluation of reports, and use of structured reports. A didactic activity index (DAI) was calculated as the sum of answers to domain-specific questions and tested for relation to demographic variables. We also asked about dictation methods and International Classification of Diseases (ICD) or Common Procedural Terminology (CPT) coding of examinations by radiologists. RESULTS: Of the 191 active radiology residencies, 151 (79%) completed the survey. Responses for hours of didactic instruction in reporting given more than a 4-year residency were distributed as follows: 0-1 = 40%, 2-4 = 46%, >4 = 14%. The percentage of resident reports formally graded was distributed as follows: 0-1 = 82%, 2-4 = 8%, >4 = 10%. The extent to which faculty-designed, structured reports were used by residents was distributed as follows: none = 16%, minimal = 25%, few = 17%, some = 33%, most = 9%. The DAI was normally distributed with a mean of 14.8 and a standard deviation of 2.4. Military programs had higher DAIs than university residencies (P = .03). There was no significant relation between any other program demographic variables and the DAI (P > .05). A substantial number of programs reported that physicians performed coding for some or most studies: ICD-9 = 30%, CPT = 26%. The dominant method for report generation was human transcription in 79% followed by speech recognition at 19%. Speech recognition penetration (departments reporting use of the technology for at least some dictation) was estimated to be 38%. CONCLUSION: In 86% of sampled radiology residencies, trainees receive no more than one hour of didactic instruction in radiology reporting per year. An aggregate measure of didactic activity about interpretative reporting was identical across all program demographic variables except that military residencies seemed to do slightly more than those located at universities.

Data Collection↗
PubMed1999

A rhoptry-protein-associated mechanism of clonal phenotypic variation in rodent malaria.

The recognition and invasion of host cells are mediated by components of the apical complex of the ookinete, sporozoite and merozoite stages of Plasmodium parasites. The paired rhoptries (organelles involved in host-cell recognition) in the apical complex contain many proteins of as-yet unknown function. In the rodent malaria agent P. yoelii yoelii, a multigene family codes for merozoite rhoptry proteins of relative molecular mass 235,000 (p235 proteins); these proteins are thought to determine the subset of erythrocytes that the parasites invade. Further support for this idea came from the identification of a region in p235 with weak but significant homology to reticulocyte-binding protein-2 of P. vivax and the demonstration that at least one p235 member binds to the erythrocyte surface membrane. Here, using single, micromanipulated P.y.yoelii parasites, we describe a new mechanism of gene expression by which the merozoites originating from a single schizont each express a distinct member of this multigene family. We propose that this new type of clonal phenotypic variation provides the parasite with a survival strategy in the mammalian host; this strategy contributes to the observed chronicity of malarial infections. This phenomenon is genetically and functionally distinct from classical antigenic variation, which is mediated by the var multigene family of P. falciparum.

Animals↗
PubMed2000

Zinc ion mediated amino acid discrimination by threonyl-tRNA synthetase.

Accurate translation of the genetic code depends on the ability of aminoacyl-tRNA synthetases to distinguish between similar amino acids. In order to investigate the basis of amino acid recognition and to understand the role played by the zinc ion present in the active site of threonyl-tRNA synthetase, we have determined the crystal structures of complexes of an active truncated form of the enzyme with a threonyl adenylate analog or threonine. The zinc ion is directly involved in threonine recognition, forming a pentacoordinate intermediate with both the amino group and the side chain hydroxyl. Amino acid activation experiments reveal that the enzyme shows no activation of isosteric valine, and activates serine at a rate 1,000-fold less than that of cognate threonine. This study demonstrates that the zinc ion is neither strictly catalytic nor structural and suggests how the zinc ion ensures that only amino acids that possess a hydroxyl group attached to the beta-position are activated.

Binding Sites↗
PubMed2005

Parental experiences of adult child death from cancer.

BACKGROUND: Although parents experience the death of adult children diagnosed with cancer, most of the literature on adult child death has examined the issues of violent death or suicide. Where death from cancer has been studied, the focus has been mainly on psychological outcomes. Little is known about parents' experiences with this phenomenon. OBJECTIVE: To understand parents' experience of having an adult child die from cancer. DESIGN: Descriptive-exploratory study utilizing semistructured interviews with parents of 10 adult children who had died of cancer. SETTING: Parents who had lost a child to cancer were interviewed in their own home or in the interviewer's office. ANALYSIS: Interviews were recorded, transcribed, and analyzed. Latent content analysis and constant comparison techniques were used to develop coding labels, which were then classified into categories and subcategories. RESULTS: Parents experienced a tension between their strong desire to "parent" their child, and their recognition that their child was an autonomous adult, sometimes with a spouse and family who took precedence over them. Parents of a dying child felt helpless, and sometimes were excluded/unacknowledged by health care professionals. To cope, they reconfigured their parent role using various strategies. After the death of their child further reconfiguring takes place, including preserving memories of the child, and talking about him/her. Continuing family and social relationships influence this stage. CONCLUSION: The conflict of wishing to "parent" a dying adult child while recognizing the child's autonomy creates uncertainty for parents at an already stressful time. Facilitating parent-child communication during the illness is important. After the death of the adult child parents may continue to face difficulty with their perceived role in society (parent and/or bereaved person), and sometimes within their family. Health care professionals should make greater efforts to include the parents of the dying adult in the circle of care. The bereavement experience of these parents is intense and long-lasting. Further research is needed regarding the ways in which health care providers can best assist parents to successfully integrate this profound loss.

Adult Children↗
PubMed1999

Computer analysis of transcription regulatory patterns in completely sequenced bacterial genomes.

Recognition of transcription regulation sites (operators) is a hard problem in computational molecular biology. In most cases, small sample size and low degree of sequence conservation preclude the construction of reliable recognition rules. We suggest an approach to this problem based on simultaneous analysis of several related genomes. It appears that as long as a gene coding for a transcription regulator is conserved in the compared bacterial genomes, the regulation of the respective group of genes (regulons) also tends to be maintained. Thus a gene can be confidently predicted to belong to a particular regulon in case not only itself, but also its orthologs in other genomes have candidate operators in the regulatory regions. This provides for a greater sensitivity of operator identification as even relatively weak signals are likely to be functionally relevant when conserved. We use this approach to analyze the purine (PurR), arginine (ArgR) and aromatic amino acid (TrpR and TyrR) regulons of Escherichia coli and Haemophilus influenzae. Candidate binding sites in regulatory regions of the respective H.influenzae genes are identified, a new family of purine transport proteins predicted to belong to the PurR regulon is described, and probable regulation of arginine transport by ArgR is demonstrated. Differences in the regulation of some orthologous genes in E.coli and H.influenzae, in particular the apparent lack of the autoregulation of the purine repressor gene in H.influenzae, are demonstrated.

Arginine↗
PubMed1995

A novel amber mutation in a beta zero-thalassaemia gene (beta 37TGG-->TAG), with direct detection by mapping the restriction fragments in amplified genomic DNA.

A novel amber mutation, a G to A substitution at the second position of codon 37 in the beta-globin gene that changes the tryptophan coding triplet (TGG) to a termination codon (TAG), was found in a Chinese beta-thalassaemia carrier. The mutant gene creates an additional Dde I recognition site and eliminates the Ava II site, so this point mutation can be directly identified by restriction enzyme analysis.

Adult↗
PubMed1998

Evidence against the double-arginine motif as the only determinant for protein translocation by a novel Sec-independent pathway in Escherichia coli.

Proteins which are synthesized with a signal peptide containing a 'double-arginine' motif may be translocated across the bacterial cytoplasmic membrane by a mechanism that is different from the known Sec and signal recognition particle pathways. The function of the double-arginine motif as a determinant for this novel pathway was studied by expressions of gene constructs coding for the high potential iron-sulfur protein (HiPIP) from Chromatium vinosum D in Escherichia coli. When the protein was produced with its original double-arginine motif-containing signal peptide, it was in part translocated into the periplasm and thereby processed, as shown by immunoblots after cell fractionation and N-terminal sequencing of purified HiPIP. Processing was not inhibited significantly by 3 mM sodium azide, indicating that translocation of HiPIP occurs by a SecA-independent pathway. Translocation of HiPIP could be altered to the SecA-dependent mode when its signal peptide was substituted by that of PelB from Erwinia carotovora. When the HiPIP double-arginine motif (SRRDAVK) was introduced into the corresponding position of the PelB signal peptide, the transport pathway remained SecA-dependent. This indicates that additional determinants are required for translocation by the Sec-independent pathway.

Adenosine Triphosphatases↗
PubMed1994

The Saccharomyces cerevisiae mutation elm4-1 facilitates pseudohyphal differentiation and interacts with a deficiency in phosphoribosylpyrophosphate synthase activity to cause constitutive pseudohyphal growth.

Saccharomyces cerevisiae mutant E124 was selected in a visual screen based on elongated cell shape. Genetic analysis showed that E124 contains two separate mutations, pps1-1 and elm4-1, each causing a distinct phenotype inherited as a single-gene trait. In rich medium, pps1-1 by itself causes increased doubling time but does not affect cell shape, whereas elm4-1 results in a moderate cell elongation phenotype but does not affect growth rate. Reconstructed elm4-1 pps1-1 double mutants display a synthetic phenotype in rich medium including extreme cell elongation and delayed cell separation, both characteristics of pseudohyphal differentiation. The elm4-1 mutation was shown to act as a dominant factor that potentiates pseudohyphal differentiation in response to general nitrogen starvation in a genetic background in which pseudohyphal growth normally does not occur. Thus, elm4-1 allows recognition of, or response to, a pseudohyphal differentiation signal that results from nitrogen limitation. PPS1 was isolated and shown to be a previously undescribed gene coding for a protein similar in amino acid sequence to phosphoribosylpyrophosphate synthase, a rate-limiting enzyme in the biosynthesis of nucleotides, histidine, and tryptophan. Thus, the pps1-1 mutation may generate a nitrogen limitation signal, which when coupled with elm4-1 results in pseudohyphal growth even in rich medium.

Amino Acid Sequence↗
PubMed2003

Evaluation of the high-resolution speech coding strategy for the Clarion CII cochlear implant system.

Four postlinguistically deafened adults were implanted with the Clarion CII cochlear implant with the HiFocus II electrode in an evaluation of performance with a new speech coding strategy (high resolution) compared with current speech coding strategies (multiple pulsatile sampler, continuous interleaved sampling, and simultaneous analog stimulation). These strategies were implemented in the Platinum speech processor from Advanced Bionics Corporation (Sylmar, CA). Postoperatively, subjects were fitted with the traditional coding strategies and over the first month were allowed to determine their strategy of choice. This strategy was used to evaluate open-set speech recognition performance at 1 month and 3 months postfitting. At 3 months postfitting, subjects were reprogrammed with the high-resolution strategy. They returned for speech recognition testing at 1 month and 3 months postfitting with this strategy. Performance was significantly better with the high-resolution strategy for all four subjects, particularly when listening to speech in background noise. This finding was in agreement with their strong preference for the high-resolution strategy, and all four patients continue to use the high-resolution strategy.

Adult↗
PubMed1999

Color discrimination, color naming and color preferences in 80-year olds.

The aim of the present study was to investigate color discrimination, color naming and color preference in a random sample of 80-year-old men and women. Knowledge of color perception in old age can be of value when using color contrast, cues and codes in the environment to promote orientation and function. The color naming test indicated that the colors white, black, yellow, red, blue and green promoted recognition to the highest degree among all subjects. A gender-related difference, in favor of women, occurred in naming five of the mixed colors. Women also used more varied color names than men. Color discrimination was easier in the red and yellow area than in the blue and green area. This result correlates positively with visual function on far sight, and negatively with diagnosis of a cataract. The preference order for seven colors put blue, green and red at the top, and brown at the bottom, hence agreeing with earlier studies, and indicating that the preference order for colors remains relatively stable also in old age. This result should be considered when designing environments for old people.

Aged↗
PubMed2005

The importance of immune gene variability (MHC) in evolutionary ecology and conservation.

Genetic studies have typically inferred the effects of human impact by documenting patterns of genetic differentiation and levels of genetic diversity among potentially isolated populations using selective neutral markers such as mitochondrial control region sequences, microsatellites or single nucleotide polymorphism (SNPs). However, evolutionary relevant and adaptive processes within and between populations can only be reflected by coding genes. In vertebrates, growing evidence suggests that genetic diversity is particularly important at the level of the major histocompatibility complex (MHC). MHC variants influence many important biological traits, including immune recognition, susceptibility to infectious and autoimmune diseases, individual odours, mating preferences, kin recognition, cooperation and pregnancy outcome. These diverse functions and characteristics place genes of the MHC among the best candidates for studies of mechanisms and significance of molecular adaptation in vertebrates. MHC variability is believed to be maintained by pathogen-driven selection, mediated either through heterozygote advantage or frequency-dependent selection. Up to now, most of our knowledge has derived from studies in humans or from model organisms under experimental, laboratory conditions. Empirical support for selective mechanisms in free-ranging animal populations in their natural environment is rare. In this review, I first introduce general information about the structure and function of MHC genes, as well as current hypotheses and concepts concerning the role of selection in the maintenance of MHC polymorphism. The evolutionary forces acting on the genetic diversity in coding and non-coding markers are compared. Then, I summarise empirical support for the functional importance of MHC variability in parasite resistance with emphasis on the evidence derived from free-ranging animal populations investigated in their natural habitat. Finally, I discuss the importance of adaptive genetic variability with respect to human impact and conservation, and implications for future studies.

Journal Article↗
PubMed1993

Co-chairman's remarks: protein designs for the specific recognition of DNA.

The selective expression of a gene is achieved through the interaction of protein transcription factors with characteristic nucleotide sequences located in the regulatory region of the gene, which is usually distinct from the coding region. These proteins contain domains which bind specifically to the DNA sites (or response elements). Some general principles in the design of these DNA-binding domains are described, followed by examples of the different structural classes discovered so far and how they recognise their binding sites.

Animals↗