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Biomedical subjects

E A Greene

Publications and source records attributed to E A Greene.

At least 19 recordsLinked to original sources
PubMed2000

RNA polymerase I transcription factor Rrn3 is functionally conserved between yeast and human.

We have cloned a human cDNA that is related to the RNA polymerase I transcription factor Rrn3 of Saccharomyces cerevisiae. The recombinant human protein displays both sequence similarity and immunological crossreactivity to yeast Rrn3 and is capable of rescuing a yeast strain carrying a disruption of the RRN3 gene in vivo. Point mutation of an amino acid that is conserved between the yeast and human proteins compromises the function of each factor, confirming that the observed sequence similarity is functionally significant. Rrn3 is the first RNA polymerase I-specific transcription factor shown to be functionally conserved between yeast and mammals, suggesting that at least one mechanism that regulates ribosomal RNA synthesis is conserved among eukaryotes.

Amino Acid Sequence↗
PubMed2000

Increased coverage of protein families with the blocks database servers.

The Blocks Database WWW (http://blocks.fhcrc.org ) and Email (blocks@blocks.fhcrc.org ) servers provide tools to search DNA and protein queries against the Blocks+ Database of multiple alignments, which represent conserved protein regions. Blocks+ nearly doubles the number of protein families included in the database by adding families from the Pfam-A, ProDom and Domo databases to those from PROSITE and PRINTS. Other new features include improved Block Searcher statistics, searching with NCBI's IMPALA program and 3D display of blocks on PDB structures.

Amino Acid Sequence↗
PubMed2000

Targeted screening for induced mutations.

With the accumulation of large-scale sequence data, emphasis in genomics has shifted from determining gene structure to testing gene function, and this relies on reverse genetic methodology. Here we explore the feasibility of screening for chemically induced mutations in target sequences in Arabidopsis thaliana. Our TILLING (Targeting Induced Local Lesions IN Genomes) method combines the efficiency of ethyl methanesulfonate (EMS)-induced mutagenesis with the ability of denaturing high-performance liquid chromatography (DHPLC) to detect base pair changes by heteroduplex analysis. Importantly, this method generates a wide range of mutant alleles, is fast and automatable, and is applicable to any organism that can be chemically mutagenized.

Alleles↗
PubMed1998

MtENOD16 and 20 are members of a family of phytocyanin-related early nodulins.

We have identified two single-copy genes from the model legume. Medicago truncatula (MtENOD16 and 20) whose expression can be correlated with early stages of root nodulation and whose predicted coding sequences are partially homologous to both pea/vetch ENOD5 and soybean N315/ENOD55. Database searching and sequence alignment have defined the encoded early nodulins as a distinct sub-family of phytocyanin-related proteins, although the absence of key ligands implies that they are unlikely to bind copper. Molecular modelling based on known phytocyanin structure has been used to predict the 3-dimensional conformation of the principle globular domain of MtENOD16/20. Additional structural features common to both early nodulin and phytocyanin precursors include an N-terminal transit peptide, a highly variable (hydroxy)proline-rich sequence which probably undergoes extensive post-translational modification, and a hydrophobic C-terminal tail.

Amino Acid Sequence↗
PubMed1997

Gene families: the taxonomy of protein paralogs and chimeras.

Ancient duplications and rearrangements of protein-coding segments have resulted in complex gene family relationships. Duplications can be tandem or dispersed and can involve entire coding regions or modules that correspond to folded protein domains. As a result, gene products may acquire new specificities, altered recognition properties, or modified functions. Extreme proliferation of some families within an organism, perhaps at the expense of other families, may correspond to functional innovations during evolution. The underlying processes are still at work, and the large fraction of human and other genomes consisting of transposable elements may be a manifestation of the evolutionary benefits of genomic flexibility.

Amino Acid Sequence↗
PubMed1997

XDOM, a graphical tool to analyse domain arrangements in any set of protein sequences.

MOTIVATION: To extract the maximum possible information from a set of protein sequences, its modular organization must be known and clearly displayed. This is important both for structural and functional analysis. RESULTS: This paper presents an algorithm and a graphical interface called XDOM which performs a systematic analysis of the modular organization of any set of protein sequences. The algorithm is an automatic method to identify putative domains from sequence comparisons. The graphical tool displays the proteins as a set of linked boxes, corresponding to its domains. The method has been tested on a family of bacterial proteins and on whole genomes. It is currently applied to the complete SWISS-PROT database to build the PRODOM database. AVAILABILITY: XDOM is available free of charge by anonymous ftp:¿¿ftp://ftp.toulouse.inra.fr/pub/xdom¿ ¿. The ProDom database can be consulted at ¿¿http://protein.toulouse.inra.fr/prodom.html¿¿.

Algorithms↗
PubMed1996

The Spo0A protein of Bacillus subtilis inhibits transcription of the abrB gene without preventing binding of the polymerase to the promoter.

Repression of transcription of the abrB gene is essential to expression of many of the postexponential genes in Bacillus. The repression is due to the activity of the response regulator protein Spo0A. We have used in vitro transcription and DNase I and hydroxyl radical footprinting to explore the mechanism of transcription inhibition. Spo0A binds to specific DNA sequences (0A boxes), and two such boxes are found downstream of the tandem promoters for the abrB gene. The data indicate that both RNA polymerase and Spo0A bind simultaneously to a DNA fragment containing the promoters and the 0A boxes. The Spo0A prevents the polymerase from inducing DNA strand denaturation at the promoter for the abrB gene.

Bacillus subtilis↗
PubMed1994

Mutants of Agrobacterium VirA that activate vir gene expression in the absence of the inducer acetosyringone.

In the presence of inducer molecules produced by wounded plants, the VirA/VirG two-component positive regulatory system of Agrobacterium tumefaciens initiates transcription of virulence genes required for crown gall tumor formation. Exactly how this system enables the bacterium to respond to an environmental signal is not known, but phosphorylation of VirA and VirG plays a role. To analyze further the function of VirA, we chemically mutagenized the virA gene. Two mutants that activate vir transcription without the plant inducer acetosyringone were found; these mutants alter VirA function by distinct mechanisms. One mutant functions entirely independently of acetosyringone, whereas the activity of the second mutant is enhanced by acetosyringone. Both mutants function best at acid pH, but respond differently to specific monosaccharides that stimulate induction by wild-type VirA. Both mutant phenotypes are dominant over wild-type VirA, and both need the conserved histidine at the autophosphorylation site for strong inducer-independent vir transcription.

Acetophenones↗
PubMed1993

Technical note: equine skeletal preservation techniques to enhance teaching effectiveness.

When the decision was made to euthanatize an acutely laminitic Thoroughbred broodmare, graduate students from the Department of Animal Sciences and Industry reconstructed the skeleton for use as a teaching tool. The reproductive and gastrointestinal tracts were removed and preserved in formalin. The hide, muscle, tendons, ligaments, and organs were removed, and the bones were boiled in water for > or = 48 h to remove all remaining tissue. After boiling, the bones were soaked in gasoline to remove fat from the marrow cavities and then soaked in a bleach/detergent mixture as a final cleaning step. The bones were allowed to dry for several weeks, then a semi-gloss clear lacquer was applied to aid in preservation. The bones were connected with 17-gauge wire and supported by two 1.91-cm galvanized steel rods on a mobile platform. The vertebral column was aligned on flexible copper tube with a 1.27-cm diameter. Additional support was provided for the head and neck by aluminum and steel rods extending from the front support. The final product is a complete, mobile skeleton that will be used as a teaching aid in equine classes. The skeleton serves a function for all levels of the cognitive learning domain. Examples of applications include memorization, identification, and location of bones; use in case studies for synthesis and demonstration of brainstorming efforts; and evaluation of joint ailments for more advanced levels of learning.

Animal Husbandry↗
PubMed1991

Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells.

The presence of desmin was characterized in cultured rat and bovine satellite cells and its potential usefulness as a marker for identifying satellite cells in vitro was evaluated. In primary cultures, positive immunohistochemical staining for desmin and skeletal muscle myosin was observed in rat and bovine myotubes. A small number of mononucleated cells (20% of rat satellite cells and 5% of bovine satellite cells) were myosin-positive, indicative of post-mitotic differentiated myocytes. In bovine satellite cell cultures 13% of the mononucleated cells were desmin-positive, while 84% of the mononucleated cells in rat satellite cell cultures were desmin-positive. Rat satellite cell mass cultures and bovine satellite cell clonal density cultures were pulsed with 3H-thymidine, and autoradiographic data revealed that greater than 94% of dividing rat cells were desmin-positive, suggesting that desmin is synthesized in proliferating rat satellite cells. However, no desmin was seen in cells that incorporated labeled thymidine in bovine satellite cell clones. Analysis of clonal density cultures revealed that only 14% of the mononucleated cells in bovine satellite cell colonies were desmin-positive, whereas 98% of the cells in rat satellite cell colonies were desmin-positive. Fibroblast colonies from both species were desmin-negative. In order to further examine the relationship between satellite cell differentiation and desmin expression, 5-bromo-2'-deoxyuridine (BrdU) was added to culture medium at the time of plating to inhibit differentiation. Fusion was inhibited in rat and bovine cultures, and cells continued to divide. Very few desmin-positive cells were found in bovine cultures, but greater than 90% of the cells in rat cultures stained positive for desmin. The presence of desmin and sarcomeric myosin was also evaluated in regenerating rat tibialis anterior five days after bupivacaine injection. In regenerating areas of the muscle many desmin-positive cells were present, and only a few cells stained positive for skeletal muscle myosin. Application of desmin staining to rat satellite cell growth assays indicated that rat satellite cells cultured in serum-containing medium were contaminated with fibroblasts at levels that ranged from approximately 5% in 24 hr cultures to 15% in mature cultures. In defined medium 4 day cultures contain approximately 95% to 98% desmin-positive satellite cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Growth factor regulation of bovine satellite cell growth in vitro.

This study examined the effects of basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF-beta) on the proliferation and differentiation of primary bovine satellite cells (BSC) in vitro. Individually, these three factors had the following effects on satellite cells: bFGF stimulated proliferation (P less than .01) but inhibited differentiation (P less than .05); IGF-I had no effect on proliferation but stimulated differentiation (P less than .01); and TGF-beta inhibited both proliferation and differentiation (P less than .01). When combined, the following effects were observed: maximum stimulation of proliferation (P less than .01) occurred in the presence of bFGF and IGF-I and differentiation was not stimulated; TGF-beta and bFGF continued to inhibit differentiation (P less than .01), but in the presence of bFGF, TGF-beta stimulated proliferation (P less than .01). No stimulation was observed in the presence of TGF-beta and IGF-I. Bovine satellite cells respond to these three growth factors that have been shown to regulate the activity of other myogenic cells, and in most instances, the responses among cells from various species are similar. These experiments indicate that the interactions of growth factors may be critical in regulating bovine satellite cell activity.

Animals↗
PubMed1983

A covalently cross-linked matrix in skeletal muscle fibers.

When skeletal, cardiac, and smooth muscle is exhaustively extracted with a protein-unfolding reagent such as 6 M guanidine HCl and a disulfide-reducing reagent such as 5% beta-mercaptoethanol, a tissue ghost remains intact and retains the characteristic shape and dimensions of the tissue before extraction. In the case of chicken pectoral muscle, the tissue ghost contains 1% of the original muscle proteins. Guanidine HCl extraction followed by collagenase treatment of glycerol-extracted chicken pectoral muscle releases a clean preparation of elongated structures containing 0.2% of the original protein and representing the covalently cross-linked remnants of the muscle fibers. The material of these muscle fiber ghosts extends throughout the interior of the cell. Antibodies raised against the tissue ghosts of smooth muscle cross-react with glycerol extracted skeletal myofibrils, forming a banding pattern which coincides with the banding pattern observed when myofibrils are reacted with antibodies against titin. Titin, a large and soluble protein found in skeletal muscle, cross-reacts with our antigizzard antibody. However, amino acid analysis of the muscle fiber ghosts indicates that titin cannot be the only subunit of the insoluble polymer, but that one or more proteins with a very high glycine and alanine content and a very low basic and acidic amino acid content must also form part of the covalently cross-linked matrix. The possibility is presented that this matrix may be the basis of the superthin 2-3-nm filaments which have been observed in a variety of cell types.

Amino Acids↗
PubMed1975

Potassium influx in the frog atrium during the cardiac cycle.

A method for measuring inwardly directed transmembrane tracer flow during the cardiac cycle was developed and applied to a study of 42K influx in frog atrial trabeculae. A fine frog atrial fiber was suspended in a stream of nonisotopic perfusate into which a smaller tracer-containing bolus could be injected, subjecting the fiber to a brief, controlled exposure to the tracer at any desired point in the cardiac cycle. In an experiment, the tissue was exposed to a fixed number of radioactive pulses at a selected point in the cardiac cycle; a brief flush with nonradioactive perfusate removed ambient and extracellular label and an extended wash and removed the remaining intracellular tracer for radioassay. The same procedure was repeated at different points in the cycle, and the resulting tracer uptake at each point measured the relative influx of the particular ion. In this way, a characteristic and reproducible 42K influx profile was demonstrated which exhibited a marked drop below diastolic values during the first 500 msec or so of the action potential followed by a rise and an overshoot above resting values. The time of return to the resting level was somewhat uncertain but was tentatively placed in the vicinity of rapid repolarization. We suggest that the rise and the overshoot reflect the activity of the membrane Na+, K+-adenosinetriphosphatase.

Animals↗