Parameters of polyethylene glycol-induced cell fusion and hybridization in lymphoid cell lines

Vaughan, Vicki L.; Hansen, Donald; Stadler, Janice

doi:10.1007/bf01542690

Cited by 34 publications

(11 citation statements)

References 12 publications

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“…For example, PEG was applied as a precipitating agent for crystallization of proteins, 1 a filter for osmotic ultrafiltration, 2 DNA introduction into cell 3 and cell fusion reagent, 4 and in other researches in biology. [5][6][7] PEG was also useful as separation media for protein analysis 8,9 because PEG does not specifically interact with biomaterials.…”

Section: Introductionmentioning

confidence: 99%

Basic Chromatographic Properties of Polyethylene Glycol-type, Polymer-based Monolithic Columns

2010

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Section: Introductionmentioning

confidence: 99%

Basic Chromatographic Properties of Polyethylene Glycol-type, Polymer-based Monolithic Columns

2010

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“…Cells were fused with inactivated Sendai virus as described previously (Smith et al, 1975), or using a polyethylene glycol gradient (Vaughan et al, 1976). A total of 40 clones (13 primary and 27 secondary) derived from eight separate hybrid izations between the mouse HPRT deficient RAG cell line and human long term lymphoid lines or fetal cells were isolated using the cloning method described by H am and Puck (1962) and charac terized.…”

Section: Cell Tines and Hybridization Proceduresmentioning

confidence: 99%

Assignment of the gene for human neutral alpha-glucosidase C to chromosome 15

Martiniuk¹,

Hirschhorn²,

Smith³

1980

Cytogenet Genome Res

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Human neutral α-glucosidase C (GANC) can be separated from the homologous mouse isozyme by starch gel electrophoresis at pH 6.5. A total of 40 clones (13 primary and 27 secondary) were derived from eight separate hybridization experiments between the mouse HPRT deficient RAG cell line and eight different human long term lymphoid cell lines or fetal cells. The thirteen primary clones showed 100% concordance between the expression of the human enzyme and the presence or absence of human chromosome 15. Analysis of the 27 secondary clones showed only two subclones discordant for segregation of human GANC and enzyme markers for 15. The two apparently discordant clones for human GANC were both derived from the same RAG × human fetal lung primary clone, and both lacked GANC activity, while retaining a 15. Since human GANC is polymorphic with a null allele at high frequency (Martiniuk and Hirschhorn, 1980), it is possible that these subclones carried one chromosome with a null allele for GANC. Alternatively there could been an undetected chromosome break between the GANC locus and the loci of the marker enzymes. Whatever the reason for the two apparently discordant subclones, combined data from all 40 clones show 95% concordant segregation for human GANC and No. 15.

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“…Three human fibroblast lines (from fetal lung, liver, and kidney) were hybridized to the hypoxanthine phosphoribosyltransferase-negative (HPRT-) mouse RAG cell line (19). Parental cells were first fused by centrifugation through a 7-50% polyethylene glycol (Mr 7,500) gradient (20) and heterokaryons were cultured in hypoxanthine/aminopterin/thymine selective medium (21).…”

Section: Methodsmentioning

confidence: 99%

Regional assignment of the structural gene for human alpha-L-iduronidase.

Schuchman¹,

Astrin²,

Aula³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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The structural gene encoding human a-Liduronidase has been assigned to chromosome 22 by using inmunologic, electrophoretic, and somatic cell hybridization techniques. Polyclonal rabbit antibodies raised against purified human low-uptake a-L-iduronidase were used to discriminate the human and murine isozymes by a sensitive immunoprecipitation assay. The human chromosome constitution of each clone was determined by cytogenetic and enzyme marker electrophoretic techniques. In 65 human (fibroblast)-mouse (RAG) somatic cell hybrids (from four independent fusions), the expression of human a-L-iduronidase was 100% concordant with the presence of human chromosome 22; the assignment was confirmed by the demonstration of the human enzyme in tertiary somatic cell hybrids containing only chromosome 22. Further verification of the gene assignment was made by detection of the human enzyme in tertiary chromosome 22 positive hybrids by Ouchterlony immunodiffusion and rocket immunoelectrophoretic experiments with polyclonal anti-human a-L-iduronidase antibodies that were monospecific for the human enzyme. Expression of human enzymatic activity in chromosome 22 positive hybrid lines was markedly reduced; for example, a tertiary hybrid (R-G21-J-15), which contained an average of 1.7 chromosome 22s per cell, only had about 15% of the activity detected in normal diploid fibroblasts. Immunologic studies suggested that the reduced expression was due to abnormal post-translational processing or aggregation (or both) of the human and murine isozymes in these hybrids. Regional assignment of the human structural gene to 22pter -* qll was accomplished by gene dosage studies using diploid human fibroblast lines that were partially monosomic or trisomic for chromosome 22.a-L-Iduronidase (a-L-iduronide iduronohydrolase, EC 3.2.1.76) is a lysosomal hydrolase required for the degradation of the mammalian glycosaminoglycans, dermatan sulfate and heparan sulfate (1-3). Fibroblast endocytosis studies have revealed that the normal human enzyme exists in two major forms, termed high uptake and low uptake (4-7). Both enzyme forms are monomeric glycoproteins whose respective physical and kinetic properties have been determined (8-12). Mucopolysaccharide storage diseases resulting from the deficient activity of this enzyme have been described in human (1, 2, 13-15), feline (16), and canine (17) species. The human disorders, Hurler, Scheie, and the Hurler-Scheie compound, have been of particular interest to geneticists because they represent phenotypically diverse diseases that presumably result from different, recessive mutations in the structural gene encoding this enzyme (15).During the past decade, a-L-iduronidase also has served as a prototype for studies of the receptor-mediated endocytosis (4-7) and maturation (18) of lysosomal enzymes. However, despite the fact that this hydrolase has occupied a central role in human cell biology and genetics, the chromosomal location of its structural gene has not been determined. In this communication,...

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Parameters of polyethylene glycol-induced cell fusion and hybridization in lymphoid cell lines

Cited by 34 publications

References 12 publications

Basic Chromatographic Properties of Polyethylene Glycol-type, Polymer-based Monolithic Columns

Basic Chromatographic Properties of Polyethylene Glycol-type, Polymer-based Monolithic Columns

Assignment of the gene for human neutral alpha-glucosidase C to chromosome 15

Regional assignment of the structural gene for human alpha-L-iduronidase.

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