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J I Garrels

Publications and source records attributed to J I Garrels.

At least 37 records · Page 2Linked to original sources
PubMed1992

Global changes in gene expression related to antibiotic synthesis in Streptomyces hygroscopicus.

Two-dimensional gel electrophoresis was used to follow changes in gene expression associated with antibiotic (bialaphos) biosynthesis in Streptomyces hygroscopicus. Cultures were pulse-labelled with [35S]-methionine before, during, and after the switch from primary to secondary metabolism in order to compare kinetic profiles of bialaphos (antibiotic) production (bap) genes during this metabolic transition. Separation of gene products on two-dimensional gels revealed that 27 were dependent on brpA for optimal expression and were activated as the culture approached stationary phase. Genes which encoded 10 brpA-dependent proteins were mapped to a 10 kb SstI fragment of the 35 kb bap gene cluster by expressing them in Streptomyces lividans using the thiostrepton-inducible tipA promoter. N-terminal amino acid sequences of two brpA-dependent proteins, obtained by direct microsequencing of protein spots excised from two-dimensional gels, identified them as gene products mapping to the same region and involved in secondary metabolic conversions of the bap pathway. The kinetics of synthesis of 16 brpA-dependent gene products were characterized using QUEST computer software. Cluster analysis performed on the kinetics of synthesis of 346 of the most highly expressed gene products of HP5-29, including 16 brpA-dependent ones, identified 75 families having distinct patterns of expression. Many brpA-dependent proteins were clustered together; 10 were found in one kinetic family. These kinetic families also included brpA-independent gene products perhaps subject to similar regulatory mechanisms and thus possibly involved in bialaphos biosynthesis. The activation/derepression of bap expression took place as cultures approached stationary phase and was temporally related to synthesis of ppGpp.

Amino Acid Sequence↗
PubMed1992

Analysis of embryonic mouse development: construction of a high-resolution, two-dimensional gel protein database.

Numerous studies have revealed stage specific alterations in protein synthesis that occur in mouse embryos. A thorough analysis of these changes has been hampered by limitations in the ability to resolve individual proteins, the ability to accurately quantify and manage data from two-dimensional gel images, and by variation in the staging of the embryos used. To learn more of the changes in protein synthesis that occur during early development, we constructed a protein database for the mouse embryo using the QUEST system of high-resolution, two-dimensional gel electrophoresis and computerized gel image analysis (Garrels, 1989, J. Biol. Chem., 264:526). Synchronous cohorts of embryos were labeled at 3 h intervals throughout the entire preimplantation period from fertilization to blastocyst stage in order to characterize in detail the changes in protein synthesis pattern that occur during normal preimplantation development. Additional samples were prepared from early post-implantation embryos, isolated inner cell mass and trophoblast cells, and cultured embryonic stem cells. These provide the means for identifying cell and tissue specific proteins and for characterizing changes in protein synthesis that accompany early cellular differentiation in the embryo. We present here a description of the mouse embryo database and discuss its potential usefulness to the study of mammalian embryogenesis.

Animals↗
PubMed1991

Quantitative analysis of protein synthesis in mouse embryos. I. Extensive reprogramming at the one- and two-cell stages.

A quantitative, high-resolution, two-dimensional gel protein database has been constructed for the mouse embryo. This database has been used to obtain a detailed accounting of the amount and relative timing of changes in protein synthesis that occur during the 1-, 2-, and 4-cell stages along with a description of the most prevalent developmentally regulated patterns of synthesis. We find that during these early stages of development the pattern of proteins synthesized changes to a much greater extent than previously appreciated. During the 1- and 2-cell stages, the majority (60% and 85%, respectively) of the analyzed proteins exhibit twofold or greater changes in their rates of synthesis. The periods of greatest change are the late 1-cell and mid 2-cell stages, during which an extensive remodelling of protein synthetic pattern occurs that is largely complete by 15 h following the first cleavage. Once this reprogramming is complete, very little change is observed during the late 2-cell and 4-cell stages. Cluster analysis of individual protein synthesis patterns reveals a limited number of coordinately regulated protein sets that are responsible for most of the changes observed during the 1- and 2-cell stages. During the 2-cell stage, one third of the proteins increase by an average of fivefold, another third decrease by an average of sevenfold, and 10% undergo transient changes in rates of synthesis. These patterns reflect the switch from zygotic to maternal mRNA utilization following transcriptional activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Quantitative exploration of the REF52 protein database: cluster analysis reveals the major protein expression profiles in responses to growth regulation, serum stimulation, and viral transformation.

Quantitative protein databases reveal the response of cells to experimental variables, such as exposure to growth factors or transfection with a transforming gene. The nature of the response depends on the type of cell and its internal state at the time of the stimulus. By constructing a protein database to study a given cell line, we can better understand the differentiated state of the cell, the growth regulatory mechanisms it employs, the particular mechanisms it uses to cope with its environment, and the ways these mechanisms may have been compromised through mutation or transformation. The REF52 database is a quantitative database designed to study growth control and transformation in a well-defined family of normal and transformed rat cell lines. The database, which has been described and analyzed elsewhere (J. I. Garrels and B. R. Franza, J. Biol. Chem. 1989, 264, 5283-5298 and J. I. Garrels and B. R. Franza, J. Biol. Chem. 1989, 264, 5299-5312) is further explored here using cluster analysis. This method reveals the most common protein expression profiles for each series of two-dimensional gels without requiring any prior hypothesis or queries on the part of the investigator. This study reveals, for each experiment, large and small clusters of protein expression profiles, most of which have readily apparent biological meaning. For example, large clusters of proteins induced or repressed during growth to confluence have been revealed, and several clusters of transformation-sensitive proteins reveal differential effects of transformation by DNA- and RNA-tumor viruses. This analysis extends our earlier quantitative explorations of the REF52 protein database and helps to show how such a database can be used to provide context and guidance for molecular studies of regulation in a given cell system.

Algorithms↗
PubMed1989

Identification, characterization, and purification of two mammalian stress proteins present in mitochondria, grp 75, a member of the hsp 70 family and hsp 58, a homolog of the bacterial groEL protein.

We describe the identification, characterization, and purification of two members of the mammalian stress protein family, both of which are shown to be components of the mitochondria. The first, with an apparent mass of 58,000 daltons, is a constitutive protein whose synthesis increases in cells exposed to elevated temperatures and/or amino acid analogs and is therefore referred to as heat shock protein hsp 58 (hsp 58). The second, with an apparent mass of 75,000 daltons, is also a constitutive protein whose synthesis is increased in cells following glucose deprivation or exposure to either a calcium ionophore or 2-deoxyglucose and therefore represents a member of the so-called glucose-regulated proteins (grp 75). In cells treated with the potassium ionophore, nonactin, both hsp 58 and grp 75 were observed to accumulate in precursor form. Since nonactin has been reported to specifically inhibit the processing of cytoplasmic precursor proteins destined for the mitochondria, we investigated whether mature hsp 58 and grp 75 were components of the mitochondria. Mitochondria were isolated from rat liver and shown to contain both hsp 58 and grp 75. Indirect immunofluorescence using antibodies specific to either hsp 58 or grp 75 confirmed their presence within mitochondria. Proteolytic digestion experiments with intact mitochondria indicated that both proteins were not accessible to external proteolytic attack, suggesting that they are not exposed on the cytoplasmic face of the outer membrane. Based on a variety of biochemical and immunological criteria, grp 75 is shown to be a member of the hsp 70 family of stress proteins, while hsp 58 represents the mammalian equivalent of the bacterial groEL protein. Procedures for the purification of both hsp 58 and grp 75 are presented. The possible biochemical role of these two mitochondrial stress proteins is discussed in relation to the known biochemical function of their related stress protein counterparts.

Animals↗
PubMed1989

Transformation-sensitive and growth-related changes of protein synthesis in REF52 cells. A two-dimensional gel analysis of SV40-, adenovirus-, and Kirsten murine sarcoma virus-transformed rat cells using the REF52 protein database.

Two-dimensional gels of normal and virally transformed REF52 cells have been quantified and compared using the QUEST system for construction and analysis of protein databases. The REF52 protein map is based on more than 1600 high quality spots, and the relative amounts of these proteins are studied in 79 gels representing 12 major experiments. REF52 cells transformed by SV40, adenovirus, and Kirsten murine sarcoma virus (KiMSV) are compared to normal REF52 cells at several stages of growth from low density to confluence and after refeeding confluent cells. In addition, early (1-4 h) and late (21-24 h) responses to serum stimulation were measured in normal, SV40-and adenovirus-transformed cells. The database has been analyzed with respect to 1) known marker proteins and protein sets, 2) global comparison of protein patterns, and 3) selection of unknown spots which have interesting patterns of regulation. For the marker proteins, which include the tropomyosin family and the proliferation-sensitive nuclear antigen, new aspects of regulation by growth and transformation have been revealed. Proliferation-sensitive nuclear antigen, a protein known to be involved in DNA synthesis, is growth-regulated in normal cells and overexpressed in some SV40- and adenovirus-transformed cells. Global comparisons reveal no overall correlation between growth-regulated changes and transformation-induced changes; however, a set of 26 coregulated proteins, including proliferation-sensitive nuclear antigen, was found to be overexpressed in REF52 cells transformed by SV40 or adenovirus. These proteins are synthesized at rates that correlate with the rate of cell proliferation in REF52 and Kirsten murine sarcoma virus-transformed cells but, in SV40- and adenovirus-transformed cells, these proteins are synthesized at high levels independent of the rate of growth. These data suggest that the transforming proteins of SV40 and adenovirus share a function that results in deregulation of the genes coding for a class of cell cycle-regulated proteins.

Adenoviridae↗
PubMed1989

The QUEST system for quantitative analysis of two-dimensional gels.

The strategies and methods used by the QUEST system for two-dimensional gel analysis are described, and the performance of the system is evaluated. Radiolabeled proteins, resolved on two-dimensional gels and detected using calibrated exposures to film, are quantified in units of disintegrations per minute or as a fraction of the total protein radioactivity applied to the gel. Spot quantitation and resolution of overlapping spots is performed by two-dimensional gaussian fitting. Pattern matching is carried out for groups of gels called matchsets, and within each matchset every gel is matched to every other gel. During the matching process, spots are automatically added to each pattern at positions where unmatched spots were detected in other patterns. This results in enhanced accuracy for both spot detection and for matching. The spot fitting procedure is repeated after matching. Tests show that up to 97% of spots in each pattern can be matched and that fewer than 1% of the spots are matched inconsistently. Approximately 2000 proteins are detected from typical gels. Of these 1600 are high quality spots. Tests to measure the coefficient of variation of spot quantitation versus spot quality show that the average coefficient of variation for high quality spots is 21%. The intensities of the detected proteins range from 4 to 20,000 ppm of total protein synthesis. The QUEST analysis system has been used to build a quantitative database for the proteins of normal and transformed REF52 cells, as presented in the accompanying reports (Garrels, J., and Franza, B. R., Jr. (1989) J. Biol. Chem. 264, 5283-5298, 5299-5312).

Algorithms↗
PubMed1989

The REF52 protein database. Methods of database construction and analysis using the QUEST system and characterizations of protein patterns from proliferating and quiescent REF52 cells.

The construction and analysis of protein databases using the QUEST system is described, and the REF52 protein database is presented. A protein database provides the means to store and compare quantitative and descriptive data for up to 2000 proteins from many experiments that employ computer-analyzed two-dimensional gel electrophoresis. The QUEST system provides the tools to manage, analyze, and communicate these data. The REF52 database contains experiments with normal and transformed rat cell lines. In this report, many of the proteins on the REF52 map are identified by name, by subcellular localization, and by mode of post-translational modification. The quantitative experiments analyzed and compared here include 1) a study of the quantitative reproducibility of the analysis system, 2) a study of the clonal reproducibility of REF52 cells, 3) a study of growth-related changes in REF52 cells, and 4) a study of the effects of labeling cells for varying lengths of time. Of the proteins analyzed from REF52 cells, 10% are nuclear, 6% are phosphoproteins, and 4% are mannose-labeled glycoproteins. The mannose-labeled proteins are more prominent in patterns from quiescent cells, while the synthesis of cytoskeletal proteins is generally repressed at quiescence. A small set of proteins, selected for elevated rates of synthesis is generally repressed at quiescence. A small set of proteins, selected for elevated rates of synthesis in quiescent versus proliferating cells includes one of the tropomyosin isoforms, a myosin light chain isoform, and several prominent glycoproteins. These proteins are thought to be characteristic of the differentiated state of untransformed REF52 cells. Proteins induced early versus late after refeeding quiescent cells show very different patterns of growth regulation. These studies lay the foundations of the REF52 database and provide information needed to interpret the experiments with transformed REF52 cells, which are reported in the accompanying paper (Garrels, J., and Franza, B. R., Jr. (1989) J. Biol. Chem. 264, 5299-5312).

Animals↗
PubMed1989

Enhancement of major histocompatibility class I protein synthesis by DNA damage in cultured human fibroblasts and keratinocytes.

Exposure of primary human fibroblasts or simian virus 40-transformed human keratinocytes to several different classes of DNA damage, including UV light C (254 nm), resulted in a rapid increase in the expression of human major histocompatibility class I (MHC-I) proteins. MHC-I induction was also detected after exposure to low doses of the protein synthesis inhibitor cycloheximide, suggesting that MHC-I induction by DNA damage may be a component in a derepressible cellular SOS pathway.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

In vitro establishment is not a sufficient prerequisite for transformation by activated ras oncogenes.

Activated ras genes transform REF52 cells only at low frequencies and adenovirus early region 1A collaborates with ras oncogenes to convert REF52 cells to a tumorigenic phenotype. While failure to transform did not result from an absence of ras gene expression, E1A appeared to enhance expression of transfected ras genes by approximately tenfold. However, enhanced ras expression alone does not account for collaboration by E1A since overexpression of T24 Ha-ras p21 induced morphological crisis and cell growth arrest rather than stable transformation. These results indicate that E1A contributes complementing biochemical activities that enable ras genes to transform REF52 and suggest that the role of E1A in primary cell transformation may extend beyond facilitating in vitro establishment.

Adenoviridae↗
PubMed1985

Characterization and purification of lupus antigen La, and RNA-binding protein.

HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.

Antigens↗
PubMed1984

Cell surface-associated proteins which bind native type IV collagen or gelatin.

Gelatin coupled to Sepharose has been used to isolate [35S]methionine-labeled polypeptides of Mr = 47,000, 56,000, 62,000, and 65,000 from the 12,000 X g supernatant of detergent extracts of mouse embryo parietal endoderm cells. The polypeptides can also be recovered from various established cell lines which synthesize type IV procollagen, and in these cells the Mr = 47,000 polypeptide is the major gelatin-binding component. Several lines of evidence, including the results of continuous labeling and pulse-chase experiments, show that the polypeptides are not derived by proteolytic cleavage of larger precursors and are distinct from fragments of fibronectin. The Mr = 47,000, 62,000, and 65,000 polypeptides all contain N-linked oligosaccharide side chains, as judged by their labeling with [3H]mannose and their sensitivity to tunicamycin. The Mr = 62,000 and 65,000 polypeptides can be resolved by two-dimensional gel electrophoresis into species with different isoelectric points, and the Mr = 47,000 has a pI of 7.5-8.0. None of the gelatin-binding polypeptides appear to accumulate in the culture medium, and the Mr = 47,000, 62,000, and 65,000 species are labeled in a lactoperoxidase-catalyzed iodination of intact cells, suggesting that they are associated with the cell surface. Only the Mr = 47,000 glycoprotein binds to native type IV collagen. Possible functions for these surface components in vivo are discussed.

Animals↗
PubMed1983

Biochemical characterization of the mammalian stress proteins and identification of two stress proteins as glucose- and Ca2+-ionophore-regulated proteins.

Biochemical properties of the heat shock or stress proteins of mammalian cells have been investigated using two-dimensional gel electrophoresis and immunological techniques. Of the major mammalian stress proteins (Mr = 72,000, 73,000, and 90,000) and minor stress proteins (Mr = 80,000, 100,000, and 110,000), the 80- and 90-kDa proteins were found to be phosphoproteins in all cell types examined. The 100-kDa protein was found to incorporate phosphate in only some cell types examined. In studies of the metabolic incorporation of mannose into the stress proteins, only the 100-kDa protein was found to be a glycoprotein. Two of the stress proteins, the 80- and 100-kDa species, were found to be identical with the proteins induced in cells grown in the absence of glucose (i.e. the "glucose-regulated proteins"). These same two proteins also were induced in cells treated with the calcium ionophore A23187. To begin examining the intracellular location of these multiregulated proteins, immunofluorescence microscopy studies were carried out using a monoclonal antibody against the 100-kDa stress protein. The antigen was localized primarily with the Golgi apparatus and less prominently with the plasma membrane and nucleus. Heat shock treatment resulted in an increased number of the cells exhibiting a nuclear location of 100 kDa.

Animals↗
PubMed1983

TOr1 is a novel, variant form of mouse chromosome 17 with a deletion in a partial t haplotype.

Moutier discovered, in a mouse from a noninbred Swiss/Orleans laboratory stock, a spontaneous dominant mutation which mapped to the T locus, and which was named TOr1. Genetic analyses indicated that TOr1 was not a simple mutation at one locus, but rather a deletion over a 3-centimorgan region of chromosome 17 that included both T and quaking (qk). Further experiments reported by Erickson et al., and a more comprehensive study by Hammerberg, have demonstrated that TOr1 is associated with recessive genetic properties affecting sperm function, characteristic of the proximal region of complete t haplotypes. These results were interpreted as evidence for the location of proximal t haplotype 'sperm factors' within the region deleted by TOr1. We now provide conclusive biochemical and genetic evidence that the 'TOr1 haplotype' is inseparably associated with a chromosomal region derived from a naturally occurring mouse t haplotype. Hence, it is likely that the t haplotype properties of TOr1 are a consequence not of the deletion itself, but of closely linked mutant t haplotype genes.

Animals↗