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Hereditary Spherocytosis and Hereditary Elliptocytosis: Aberrant Protein Sorting during Erythroblast Enucleation

Description: During erythroblast enucleation, membrane proteins distribute between extruded nuclei and reticulocytes. In hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), deficiencies of membrane proteins, in addition to those encoded by the mutant gene, occur. Elliptocytes, resulting from protein 4.1R gene mutations, lack not only 4.1R but also glycophorin C, which links the cytoskeleton and bilayer. In HS resulting from ankyrin-1 mutations, band 3, Rh-associated antigen, and glycophorin A are deficient. The current study was undertaken to explore whether aberrant protein sorting, during enucleation, creates these membrane-spanning protein deficiencies. We found that although glycophorin C sorts to reticulocytes normally, it distributes to nuclei in 4.1R-deficient HE cells. Further, glycophorin A and Rh-associated antigen, which normally partition predominantly to reticulocytes, distribute to both nuclei and reticulocytes in an ankyrin-1-deficient murine model of HS. We conclude that aberrant protein sorting is one mechanistic basis for protein deficiencies in HE and HS.
Date: February 8, 2010
Creator: Salomao, Marcela; Chen, Ke; Villalobos, Jonathan; Mohandas, Narla; An, Xiuli & Chasis, Joel Anne
Partner: UNT Libraries Government Documents Department

RAD51AP2, a novel vertebrate- and meiotic-specific protein, sharesa conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51

Description: Many interacting proteins regulate and/or assist the activities of RAD51, a recombinase which plays a critical role in both DNA repair and meiotic recombination. Yeast two-hybrid screening of a human testis cDNA library revealed a new protein, RAD51AP2 (RAD51 Associated Protein 2), that interacts strongly with RAD51. A full-length cDNA clone predicts a novel vertebrate specific protein of 1159 residues, and the RAD51AP2 transcript was observed only in meiotic tissue (i.e. adult testis and fetal ovary), suggesting a meiotic-specific function for RAD51AP2. In HEK293 cells the interaction of RAD51 with an ectopically-expressed recombinant large fragment of RAD51AP2 requires the C-terminal 57 residues of RAD51AP2. This RAD51-binding region shows 81% homology to the C-terminus of RAD51AP1/PIR51, an otherwise totally unrelated RAD51-binding partner that is ubiquitously expressed. Analyses using truncations and point mutations in both RAD51AP1 and RAD51AP2 demonstrate that these proteins use the same structural motif for RAD51 binding. RAD54 shares some homology with this RAD51-binding motif, but this homologous region plays only an accessory role to the adjacent main RAD51-interacting region, which has been narrowed here to 40 amino acids. A novel protein, RAD51AP2, has been discovered that interacts with RAD51 through a C-terminal motif also present in RAD51AP1.
Date: July 25, 2006
Creator: Kovalenko, Oleg V.; Wiese, Claudia & Schild, David
Partner: UNT Libraries Government Documents Department

From photons to protons in the photocycle of bacterial reaction center

Description: The detailed knowledge of the atomic coordinates of the bacterial reaction center (RC) has permitted a close scrutiny of structure/function relationships not only of the quinones but of the protein itself with its internal water structure. Protonatable groups were identified as intrinsic part of the redox reactions, providing charge compensation and forming channels for the movement of hydrogen ions to QB2-. The nature and position of these groups give rise to electrostatic profiles that determine the kinetics and energetics of proton transport. Fine tuning or dramatic variations of protein delivery pathways can adapt the photocycle to changes in bulk phase pH value, buffering capacities and primary structure of the RC.
Date: December 31, 1995
Creator: Maroti, P., Osvath, S., Tapai, C., Hanson, D.K.
Partner: UNT Libraries Government Documents Department

Disability rights in dialogue with clinical genetics conference, May 31 to June 2, 1996

Description: The issue of prenatal diagnosis and selective abortion has been hotly debated in the medical, genetic counselling, feminist, parents, disability rights and bio-ethics literature, each of the various positions critiquing each other. People from the disability rights community in particular have began to articulate a critical view of the practice of widespread prenatal diagnosis with intent to abort because the pregnancy might result in a child with a disability. Unfortunately, people from the various disciplines and perspectives, such as bioethics, disability rights, feminism and so forth, by and large, have tended only to write for themselves and their colleagues. Few people have crossed disciplines to try to talk to people with other views. The rapid advances of genome research have continued to produce new prenatal tests, raising many complex ethical questions regarding the applications of prenatal testing. But the widely disparate positions of the various factions has made it difficult to move toward formulation of public policy change necessary to encompass these new genetic technologies. Genetic counselling is in the front lines of the controversial social and ethical issues arising from prenatal diagnosis, in its interface between medical science and the consumer of services. The primary intent of the conference was to invite and facilitate productive dialogue between individuals and groups of people who have traditionally not interacted as a result of their disparate views on these issues and to learn from this process, emphasizing the involvement of people with disabilities and people who work in clinical genetics.
Date: December 31, 1996
Partner: UNT Libraries Government Documents Department

Wasted (wst) mice have 3-bp deletion in the PCNA promoter

Description: Mice homozygous for the autosomal recessive wasted mutation (wst/wst) have abnormalities in T-lymphocytes and in the anterior motor neuron cells of the spinal cord, leading to sensitivity to ionizing radiation, hind limb paralysis, and immunodeficiency. This defect results in a failure to gain weight by 20 days and death at 28 days of age. Previous results from the authors` group have shown that (1) wasted mice have little if any detectable PCNA protein or mRNA in thymus, but levels in liver, brain, and other tissues are similar to those in controls; and (2) the coding region for PCNA is the same in wasted mice and in control littermates. These observations gave rise to the present study, in which the PCNA promoter was sequenced for wst/wst mice, control littermates ({center_dot}wst/+) and BCF{sub 1} (or BALB/c x C57BL/6) F{sub 1} controls. Sequence analysis revealed only one difference between wst/wst and BALB/c x C57BL/6 F{sub 1} littermates: a 3-bp deletion in the 5 foot upstream region of the PCNA gene of wasted mice that was observed on only one allele or no alleles of normal littermates. The mutated sites in PCNA promoter from two litters plus two additional wst/wst and two known wst/+ animals were screened with 8G and 11G probes, and each confirmed this pattern. The short term DNA segment encompassing the deletion was shown in gel shift experiments to bind a nuclear protein(s) present in a broad variety of cells including thymus and spleen nuclear extract from wst/wst and control mice. The mutated oligomer that was homozygous only in wst/wst mice was not able to bind the same nuclear protein(s).
Date: August 1, 1997
Creator: Paunesku, T. & Woloschak, G.E.
Partner: UNT Libraries Government Documents Department

The biology of novel animal genes: Mouse APEX gene knockout

Description: This is the final report of a one-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The controlled breeding of novel genes into mice, including the gene knockout (KO), or conversely by adding back transgenes provide powerful genetic technologies that together suffice to determine in large part the biological role(s) of novel genes. Inbred mouse remains the best understood and most useful mammalian experimental system available for tackling the biology of novel genes. The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE), is involved in a key step in the repair of spontaneous and induced AP sites in DNA. Efficient repair of these lesions is imperative to prevent the stable incorporation of mutations into the cellular genome which may lead to cell death or transformation. Loss or modulation of base excison repair activity in vivo may elevate the spontaneous mutation rate in cells, and may lead to a substantial increase in the incidence of cancer. Despite extensive biochemical analysis, however, the significance of these individual APE functions in vivo has not been elucidated. Mouse embryonic stem (ES) cells heterozygous for a deletion mutation in APE have been generated and whole animals containing the APE mutation have been derived from these ES cells. Animals homozygous for the APE null mutation die early in gestation, underscoring the biological significance of this DNA repair gene.
Date: July 1, 1997
Creator: MacInnes, M.; Altherr, M.R.; Ludwig, D.; Pedersen, R. & Mold, C.
Partner: UNT Libraries Government Documents Department

Enzyme catalysts for a biotechnology-based chemical industry. Quarterly progress report, January 1--April 1, 1998

Description: The goal of this research is to engineer enzymes to be efficient and economically attractive catalysts for the chemical industry. The author is attempting to demonstrate generally-applicable approaches to enzyme improvement as well as develop specific catalysts for potential industrial application. The research is focused on the following areas: (1) Random mutagenesis of pNB esterase: improved activity and stability; (2) Directed evolution of subtilisin E to enhance thermostability; and (3) Methods for in vitro recombination.
Date: April 20, 1998
Creator: Arnold, F.H.
Partner: UNT Libraries Government Documents Department

Human radiation studies: Remembering the early years: Oral history of radiologist Henry I. Kohn, M.D., Ph.D., conducted September 13, 1994

Description: This report is a transcript of an interview of Dr. Henry I. Kohn by representatives of the US DOE Office of Human Radiation Experiments. Dr. Kohn was selected for this interview because of the positions he held at Oak Ridge National Laboratory, University of California at San Francisco, and Harvard Medical School. Dr. Kohn discussed his remembrances of his experiences in blood chemistry of animals and patients exposed to radiation, and his remembrances of several radiobiologists.
Date: June 1, 1995
Partner: UNT Libraries Government Documents Department

An atomic view of additive mutational effects in a protein structure

Description: Substitution of a single amino acid in a protein will often lead to substantial changes in properties. If these properties could be altered in a rational way then proteins could be readily generated with functions tailored to specific uses. When amino acid substitutions are made at well-separated locations in a single protein, their effects are generally additive. Additivity of effects of amino acid substitutions is very useful because the properties of proteins with any combination of substitutions can be inferred directly from those of the proteins with single changes. It would therefore be of considerable interest to have a means of knowing whether substitutions at a particular pair of sites in a protein are likely to lead to additive effects. The structural basis for additivity of effects of mutations on protein function was examined by determining crystal structures of single and double mutants in the hydrophobic core of gene V protein. Structural effects of mutations were found to be cumulative when two mutations were made in a single protein. Additivity occurs in this case because the regions structurally affected by mutations at the two sites do not overlap even though the sites are separated by only 9 {angstrom}. Structural distortions induced by mutations in gene V protein decrease rapidly, but not isotropically, with distance from the site of mutation. It is anticipated that cases where structural and functional effects of mutations will be additive could be identified simply by examining whether the regions structurally affected by each component mutation overlap.
Date: April 1, 1996
Creator: Skinner, M.M. & Terwilliger, T.C.
Partner: UNT Libraries Government Documents Department

Project report to STB/UO, Northern New Mexico Community College two- year college initiative: Biotechnology

Description: This report summarizes the experiences gained in a project involving faculty direct undergraduate research focused on biotechnology and its applications. The biology program at Northern New Mexico Community College has been involved in screening for mutations in human DNA and has developed the ability to perform many standard and advanced molecular biology techniques. Most of these are based around the polymerase chain reaction (PCR) and include the use of single strand conformation polymorphism analysis (SSCP), denaturing gradient gel electrophoresis (DGGE) in the screening for mutant DNA molecules, and the capability to sequence PCR generated fragments of DNA using non-isotopic imaging. At Northern, these activities have a two-fold objective: (1) to bring current molecular biology techniques to the teaching laboratory, and (2) to support the training of minority undergraduates in research areas that stimulate them to pursue advanced degrees in the sciences.
Date: March 1, 1996
Partner: UNT Libraries Government Documents Department

The nature and alternate rates of the ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) oxygenation intermediate

Description: Mutant ribulose 1,5-bisphosphate (RuBP) were employed to investigate the partitioning of carbon flow between photosynthesis or photorespiration. Previous functional and structural studies implicate active site Lys329 and Glu48 or R. rubrum RuBp in promoting addition of CO2 to the RuBP-enediol. Two novel O2-dependent side products generated by the K329A and E49Q mutants provided insight into RuBP oxygenase intermediate and roles of Lys329 and Glu48 in oxygenation.
Date: December 31, 1995
Creator: Harpel, M.R.; Chen, Yuh-Ru & Hartman, F.C.
Partner: UNT Libraries Government Documents Department

Recent advances in lung cancer biology

Description: This paper provides an overview of carcinogenesis, especially as related to lung cancers. Various growth factors and their mutated forms as oncogenes are discussed with respect to gene location and their role in the oncogenic process. Finally the data is related to lung cancer induction in uranium miners and exposure to radon.
Date: December 31, 1995
Creator: Lechner, J.
Partner: UNT Libraries Government Documents Department

9. international mouse genome conference

Description: This conference was held November 12--16, 1995 in Ann Arbor, Michigan. The purpose of this conference was to provide a multidisciplinary forum for exchange of state-of-the-art information on genetic mapping in mice. This report contains abstracts of presentations, focusing on the following areas: mutation identification; comparative mapping; informatics and complex traits; mutagenesis; gene identification and new technology; and genetic and physical mapping.
Date: December 31, 1995
Partner: UNT Libraries Government Documents Department

Modified cellulose synthase gene from 'Arabidopsis thaliana' confers herbicide resistance to plants

Description: Cellulose synthase ('CS'), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.
Date: October 11, 2000
Creator: Somerville, Chris R. & Scieble, Wolf
Partner: UNT Libraries Government Documents Department

Effects of cavities in the bacterial reaction center

Description: A site-specific double mutant of Rhodobacter capsulatus, in which the large aromatic residues M208Tyr and L181Phe in the interior of the photosynthetic reaction center (RC) complex were replaced by smaller theonine residues, showed a dramatic reduction in the number of assembled complexes and was incapable of photosynthetic growth. The cavity created by the smaller side chains interferes mostly with the assembly of the complex. Phenotypic revertants were recovered in which a spontaneous second-site mutation restored photocompetence in the presence of the original site-specific mutations. In these strains, an Ala to Pro substitution in neighboring transmembrane helix (at M271) resulted in an increased yield of RC complexes. To test the hypothesis that the original phenotype was due to a cavity, other mutants were constructed where L180Phe and M207Leu were replaced with alanines that created similar-sized voids at other positions in the membrane-spanning interior. The L180Ala-M207A mutant had the same phenotype. Coupling of the above proline substitution to these new cavity mutants also resulted in photocompetant strains that carry increased levels of RC complexes. Therefore, the proline substitution at M271 serves as a global suppressor of the phenotype caused by these internal cavities.
Date: December 31, 1995
Creator: Schiffer, M.; Deng, Y.-L.; Marrufo, A. & Hanson, D.K.
Partner: UNT Libraries Government Documents Department

A molecular genetic approach to understanding eukaryotic cellulose synthesis. Final technical report, March 15, 1995--March 14, 1997

Description: In an attempt to understand eukaryotic cellulose synthesis, this research investigates the cellulose biosynthetic pathway of Dictyostelium discoideum. Attention is focused on the following: use of a newly developed method of tagged insertional mutagenesis to generate mutants in cellulose biosynthesis; and exploiting the monolayer culture system to identify differences in mRNA populations between DIF-induced (and cellulose-synthesizing) versus non-induced cells.
Date: November 1, 1997
Creator: Blanton, R. L.
Partner: UNT Libraries Government Documents Department

Regulation of chloroplast number and DNA synthesis in higher plants. Final report, August 1995--August 1996

Description: The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focused on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The research focused on the isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.
Date: June 17, 1997
Creator: Mullet, J.E.
Partner: UNT Libraries Government Documents Department

Enzyme catalysts for a biotechnology-based chemical industry. Quarterly progress report, September 29--December 28, 1997

Description: The goal of this research is to engineer enzymes to be efficient and economically attractive catalysts for the chemical industry. The author is attempting to demonstrate generally-applicable approaches to enzyme improvement as well as develop specific catalysts for potential industrial application. In this report attention is focused on random mutagenesis of pNB esterase -- improved activity and stability. The most thermostable esterases obtained by sequential random mutagenesis (6H7) and random mutagenesis plus recombination (6sF9) each contain 9 amino acid mutations and a number of silent mutations, relative to the wild-type sequence. Eight of the mutations are present in both genes, for a total of ten potentially adaptive mutations. Because several of these mutations occurred in the same generation, it is difficult to identify the mutations responsible for the increases in activity and stability. In order to aid in this identification, the thermostable genes were recombined with the wild-type gene, in hopes of removing neutral mutations. The gene from the first-generation variant, with five amino acid substitutions was also recombined with wild-type.
Date: January 15, 1998
Creator: Arnold, F.H.
Partner: UNT Libraries Government Documents Department

[Studies of the repair of radiation-induced genetic damage in Drosophila]. Annual progress report, June 1, 1989--September 1, 1990

Description: The most exciting discovery made over the past year derives from an analysis of the interaction between DNA repair and P-element transposition. A powerful new system was developed for analyzing the repair of DNA double-strand breaks. A screen was completed of mutagenized autosomes obtained from two San Francisco laboratories with the recovery of several mutants that will provide the foundation for future efforts to clone repair related genes. At the same time, strong progress has been made in the cloning and characterization of the repair-related genes mei-41 and mus209. Finally, the efforts to clone the mei-9 gene have uncovered the existence of a unsuspected feature of the system used for transposon-tagging in Drosophila. This new knowledge will aid future cloning efforts as well as those of others in the field.
Date: December 31, 1990
Partner: UNT Libraries Government Documents Department