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J S Butel

Publications and source records attributed to J S Butel.

At least 73 records · Page 4Linked to original sources
PubMed1993

p53 mutations in mouse mammary epithelial cells: instability in culture and discordant selection of mutations in vitro versus in vivo.

The phenotypes of p53 mutations found in human and murine tumors often are analyzed using a variety of transformation assays in vitro, but data have not been available to correlate in vitro effects with in vivo activities. We have assessed the effects of p53 mutations using mouse mammary epithelial cell lines which can be analyzed both in vitro and in vivo. Parental mammary epithelial cell lines (FSK series) injected into cleared mammary fat pads of syngeneic mice frequently give rise to preneoplastic lesions (HAN) which can be reestablished in culture (TM lines) to permit analysis of genetic changes important in the development of preneoplasia. Characterization of the FSK3 cell line revealed a cell population mixed with respect to p53 genotypes. One subpopulation of mutant (Ser233-234) p53-expressing cells was selected in a preneoplastic mammary outgrowth in vivo (TM3), whereas another minor population of mutant (Pro135) p53-expressing cells was selected during culturing of FSK3 cells in vitro. When FSK3 cells were subdivided and passaged in parallel in vitro, p53 genotypes continued to evolve and diverge. These findings reveal that selective pressures exerted on mammary epithelial cell populations in vivo are different from pressures present in vitro. Selective forces may vary from one cell culture environment to another. The growth advantage conferred by a specific p53 mutation appears to differ in vivo versus in vitro. We propose that caution should be exercised when attempting to correlate the effects of p53 mutations assayed in cell culture systems with the in situ phenotypes of mutant p53 in cancer.

Amino Acid Sequence↗
PubMed1992

DNA sequences similar to those of simian virus 40 in ependymomas and choroid plexus tumors of childhood.

BACKGROUND: Ependymomas and papillomas of the choroid plexus occur in early childhood. The ubiquitous human polyomaviruses, BK virus and JC virus, have been associated with the induction of these neoplasms in animal models. A related monkey polyomavirus, simian virus 40 (SV40), is highly tumorigenic in rodents and also induces choroid plexus papillomas. METHODS: We tested the possibility that polyomaviruses were associated with these tumors in humans. Tumors from 31 children--20 with choroid plexus neoplasms and 11 with ependymomas--were evaluated for the presence of polyomavirus T-antigen gene sequences by means of amplification with the polymerase chain reaction. RESULTS: Ten of the 20 choroid plexus tumors and 10 of the 11 ependymomas contained amplification products that preferentially hybridized to probes specific for SV40 viral DNA rather than BK or JC viral DNA. In two specimens, DNA sequencing demonstrated that the amplified sequence was identical to the sequence of that region of the SV40 gene. In three other specimens, amplification with SV40-specific primers revealed a 574-bp segment of the SV40 viral gene. In 7 of 11 tumors examined by immunohistochemical staining, viral T antigen was expressed in the nuclei of the neoplastic cells. CONCLUSIONS: Half of the choroid plexus tumors and most of the ependymomas that we studied contained and expressed a segment of T-antigen gene related to SV40. These results suggest that SV40 or a closely related virus may have an etiologic role in the development of these neoplasms during childhood, as in animal models.

Antigens, Viral, Tumor↗
PubMed1992

Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours.

Mutations in the p53 tumour-suppressor gene are the most frequently observed genetic lesions in human cancers. To investigate the role of the p53 gene in mammalian development and tumorigenesis, a null mutation was introduced into the gene by homologous recombination in murine embryonic stem cells. Mice homozygous for the null allele appear normal but are prone to the spontaneous development of a variety of neoplasms by 6 months of age. These observations indicate that a normal p53 gene is dispensable for embryonic development, that its absence predisposes the animal to neoplastic disease, and that an oncogenic mutant form of p53 is not obligatory for the genesis of many types of tumours.

Alleles↗
PubMed1992

p53 mutations cluster at codon 249 in hepatitis B virus-positive hepatocellular carcinomas from China.

DNA samples from 36 hepatocellular carcinoma (HCC) patients from China were screened for a specific mutation affecting codon 249 of the p53 gene, recently identified as a hotspot mutation in some HCCs. We detected the tumor-specific p53 codon 249 mutation in 21 (58%) of 36 HCCs examined. Thirteen patients with the specific codon 249 mutation had lost the remaining allele of p53, whereas the remaining eight patients appeared to have retained both copies of the gene. These results suggest that alterations of p53 may be important events in the genesis of HCCs and that point mutation may precede allele loss.

Adult↗
PubMed1992

Differential effects of the simian virus 40 early genes on mammary epithelial cell growth, morphology, and gene expression.

To study the effect of SV40 T-antigen in mammary epithelial cells, a rat beta-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.

Adenovirus Early Proteins↗
PubMed1992

Epitope mapping and conformational analysis of SV40 T-antigen deletion mutants.

Deletion mutants of SV40 T-antigen were stably expressed in BALB/c 3T3E cells and characterized in immunoprecipitation assays using T-antigen-specific monoclonal antibodies. The epitope recognized by antibody PAb602 was narrowed to T-antigen residues 230-362. Coprecipitation results were compatible with a p53-binding region between T-antigen amino acids 347 and 517. Amino terminal deletions (1-108 or 98-229) of T-antigen appeared to have pronounced effects on the conformation of distal regions of the molecule, based both on antibody binding and on association with p53.

Animals↗
PubMed1991

Hepatitis B virus integration event in human chromosome 17p near the p53 gene identifies the region of the chromosome commonly deleted in virus-positive hepatocellular carcinomas.

The development of hepatocellular carcinoma (HCC) presumably occurs in multiple steps and is influenced by numerous factors. Hepatitis B virus (HBV) is strongly associated with the development of HCC in people chronically infected with the virus, but the mechanism of viral involvement remains unclear. One possibility is that the gross chromosomal alterations frequently observed in HCC DNA at the site of HBV integration may alter the expression of important nearby cellular genes. We previously reported the cloning and characterization of a HBV insert from a Chinese HCC. The viral insert mapped to chromosome 17p11.2-12, and cellular sequences were duplicated at the site of viral integration. In the present study a DNA probe derived from cellular DNA sequences adjacent to the previously characterized HBV insert was used to analyze a set of 19 matched normal liver and HBV-positive hepatoma samples obtained from the same region of China, near Shanghai. Tumor-specific DNA changes were detected in two additional HCCs, suggesting that the small region of chromosome 17p defined by the flanking cell DNA probe is commonly altered in hepatomas. Restriction fragment length polymorphism studies demonstrated that the loss of one copy of portions of chromosome 17 occurred in 10 (53%) of the 19 patients. The loss of one allele of the p53 gene (located on chromosome 17p13) occurred in at least 6 (60%) of the 10 patients who were heterozygous at the p53 locus. As the p53 gene is known to possess tumor suppressor activity, the functional loss of this gene may be a significant step in the development of a subset of HCCs. High levels of allele loss also were detected for chromosomes 8q (4 of 9; 44%) and 16p (5 of 6; 83%) and may indicate the presence of additional cellular genes whose functional loss is important in the development of HCCs.

Carcinoma, Hepatocellular↗
PubMed1991

Investigations on etiology of Crohn's disease. Humoral immune response to stress (heat shock) proteins.

Many investigators have tried to prove a relationship between Crohn's disease and Mycobacteria. Recent evidence suggests that some autoimmune diseases may be initiated through "molecular mimicry" between mycobacterial stress protein antigens and their human homologs. We investigated whether antibody to stress proteins was more frequent in patients with Crohn's disease than controls. We used ATP binding to separate stress proteins (heat-shock-induced, de novo-synthesized, and constitutively expressed ATP-binding proteins) from crude extracts obtained from Mycobacteria and from an SV40-transformed human epithelial cell line that expresses a heat-shock protein, hsp73, as a complex with SV40 T antigen. We used immunoblots to compare sera from 34 patients with Crohn's disease, 14 with ulcerative colitis, and 14 with duodenal or gastric ulcers (noninflammatory bowel disease control patients). We found no statistically significant pattern or frequency of antibodies against single proteins or a combination of mycobacterial or human stress proteins. These observations do not support the hypothesis that a humoral immune response to stress proteins of Mycobacteria is important in the pathogenesis of Crohn's disease.

Antibody Formation↗
PubMed1991

Detection and characterization of a glycoprotein encoded by the mouse mammary tumor virus long terminal repeat gene.

Mouse mammary tumor virus (MMTV) is a retrovirus that causes mammary tumors in susceptible mice. MMTV contains a unique open reading frame (ORF) in the unique 3' region of the proviral long terminal repeat (LTR) with the potential to encode a 36-kDa protein. However, the ORF gene product has not been detected in murine mammary tissues or cell lines. We utilized the baculovirus expression vector system to generate large amounts of the ORF protein. Putative ORF gene products of 36 and 45 kDa were detected as unique proteins in extracts of insect cells infected with recombinant baculovirus (LTR-ORF BV), and the identities of these proteins as viral gene products were confirmed immunologically. Antipeptide antisera were generated in rabbits against peptides chosen from computer-predicted hydrophilic regions of the ORF coding sequence. These antisera reacted specifically by immunoprecipitation and by immunoblot with the proteins expressed in LTR-ORF BV-infected insect cells, as well as with MMTV LTR ORF in vitro translation products. Polyclonal antisera were raised against two putative ORF protein species partially purified from insect cells. These sera specifically immunoprecipitated viral protein products translated in vitro. In vitro translation of MMTV LTR ORF transcripts in the presence of canine pancreatic microsomal membranes generated a higher-molecular-weight ORF gene product, indicating that the ORF protein is modified by N-linked glycosylation. This glycosylated ORF product comigrated with the larger ORF protein species produced in infected insect cells. The gp45 product was metabolically labeled with [3H] mannose, [3H] galactose, and [3H] N-acetyl-D-glucosamine in insect cells, whereas this incorporation was inhibited in the presence of tunicamycin. Digestion of gp45 with endoglycosidase H yielded the lower-molecular-weight ORF protein p36. These observations suggest that the ORF glycoprotein contains hybrid N-linked oligosaccharides. Demonstration of the modified nature of the ORF gene product will facilitate characterization of ORF protein expression in murine tissues.

Animals↗
PubMed1991

Simian virus 40 large T antigen and p53 are microtubule-associated proteins in transformed cells.

The cellular proteins that interact with simian virus 40 large T antigen (T-ag) must be identified in order to understand T-ag effects on cellular growth control mechanisms. A protein extraction procedure utilizing single-phase concentrations of 1-butanol recovered a complex composed of T-ag, p53, and other Mr 35,000-60,000 proteins from suspension cultures of the simian virus 40-transformed mouse cell line mKSA. Partial protease mapping showed each of the associated proteins to be unique. Automated microsequence analysis of the NH2-terminal 30 amino acids of the Mr 56,000 protein purified after coprecipitating with T-ag and p53 identified it as the beta subunit of mouse tubulin. The existence of a complex containing tubulin, T-ag, and p53 was confirmed by reciprocal immunoblotting experiments. Both T-ag and p53 were coprecipitated by three different monoclonal antibodies directed against tubulin, and conversely, monoclonal antibodies specific for T-ag or p53 coprecipitated tubulin. Mixing experiments and extractions in the presence of purified tubulin indicated that the complex existed in situ prior to cell lysis. Both p53 and T-ag copurified with microtubules through two cycles of temperature-dependent disassembly and assembly. Both T-ag and p53 were localized to microtubules in the cytoplasm of mKSA cells by immunoelectron microscopy. Treatment of mKSA cells with 10 microM colchicine followed by lysis in 0.1% Nonidet P-40 resulted in increased amounts of solubilized T-ag and p53. Both T-ag and p53 were also associated with microtubules in three other simian virus 40-transformed mouse cell lines growing as monolayers, confirming the generality of the association. An interaction of T-ag and p53 with microtubules may be important in the intracellular transport of these proteins and may affect cellular signal transduction or growth control.

Amino Acid Sequence↗
PubMed1990

Influence of mammary cell differentiation on the expression of proteins encoded by endogenous BALB/c mouse mammary tumor virus genes.

The interactions between differentiation-associated cellular events in the intact mammary gland or in cultured mammary cells and the post-transcriptional activity of the endogenous mouse mammary tumor virus (MMTV) loci were investigated. The transcriptional activities of the endogenous MMTV proviruses of the BALB/c mouse strain (Mtv-6, Mtv-8 and Mtv-9) appear to be regulated differentially during pregnancy-induced mammary gland development (J.E. Knepper, D. Medina and J.S. Butel, J. Virol. 59, 518-521, 1986). Analysis of MMTV-specific proteins at various stages of mammary gland development (virgin, midpregnant, lactating, regressing) established the presence of steady-state levels of a 67,000-Mr env precursor-type polypeptide at all physiological stages. However, processing to lower-molecular-weight env-specific proteins, including a predominant 50,000-Mr species, was detected only with the transition to the functional mammary gland phenotype. The contributions of cell proliferation, cell-matrix interactions, and modulation of functional activity to the pattern of endogenous MMTV protein expression were investigated using a 3-dimensional collagen type I culture system. Growth and cell-matrix interactions (cell polarization, lumen formation) leading to formation of 3-dimensional duct-like structures were permissive for the synthesis and processing of MMTV-specific proteins; accumulation of high levels of the 50,000-Mr env-specific polypeptide was associated with the onset of the fully functional mammary cell phenotype. Expression of MMTV-specific proteins was not due to amplification of a specific cell subpopulation. The potential of the full-length Mtv-8 and Mtv-9 proviruses to be transcribed, as indicated by their methylation status, was not dramatically different between differentiated and undifferentiated mammary cells in culture. This study indicates that MMTV transcriptional activity is reflected at the protein level in mammary tissue of BALB/c mice and that viral protein synthesis and processing may serve as important markers of different physiological stages of mammary epithelial cells. These observations also suggest a general approach to the examination of potential modulatory effects of cellular interactions (cell-cell, cell-matrix or both) known to be important in various differentiated epithelial cell systems for the expression of viral genes.

Aldosterone↗
PubMed1990

Hepatitis B virus transactivator X protein is not tumorigenic in transgenic mice.

The hepatitis B virus X protein acts as a transcriptional transactivator in vitro. To elucidate possible biological effects of X protein on liver cells in vivo, we generated four lines of transgenic mice carrying the X gene open reading frame under the control of the human alpha-1-antitrypsin regulatory region. The plasmid construct used to introduce the transgene was shown to encode a 16-kDa X protein with transactivating capability. The expression of X protein was detectable in liver tissue of transgenic animals of three of the lines by immunoblot analysis; levels of expression were highest in the first month after birth of the animals. Over 80 animals from the expressing lines were examined histologically. Most transgenic mice, some of which were observed for up to 2 years, remained normal. However, a few transgenic animals developed mild focal hepatitis, nuclear pleomorphism, focal necrosis, and/or nodular hyperplasia in the liver. Increased mitotic activity of hepatocytes also was observed. We conclude that, at the level of expression achieved in these transgenic mice, the hepatitis B virus transcriptional transactivator X protein alone does not appear to mediate the development of serious liver damage or hepatocellular carcinomas.

Animals↗
PubMed1990

SV40 large T antigen directed by regulatory elements of the human alpha-1-antitrypsin gene. A transgenic mouse system that exhibits stages in liver carcinogenesis.

SV40 large T antigen (T-ag) is a potent oncogene able to transform many cell types. The human alpha-1-antitrypsin (AAT) gene encodes the major serine protease inhibitor in plasma. The 5' flanking sequence of the AAT gene contains cis-acting elements that specify the expression of reporter genes in transgenic mice. Although the AAT gene is expressed primarily in the liver, it also is expressed in epithelial cells of the stomach, kidney, intestine and pancreas. A fusion gene with the 5' flanking region of AAT driving the SV40 large T-ag gene was used to generate transgenic mice. Between 2 and 6 months of age, 6 of 7 founder mice developed hepatic neoplasms. Kidney hyperplasia (3/7) and carcinomas of the stomach (4/7) and pancreas (1/7) also were observed. Tumor cells in the different sites expressed T-ag that was detectable by immunohistochemical staining. A transgenic mouse line (1812) was established; liver tumors develop reproducibly in all offspring. Cellular changes progressing to liver tumor formation occur in discrete stages with predictable kinetics in animals of the 1812 line: (i) embryonal/fetal stage, no major histological changes; (ii) newborn to 2 weeks of age, hyperplastic livers with minor cellular changes; (iii) mice between 3 and 8 weeks of age, diffuse liver cell dysplasia without observable tumor nodules; (iv) mice 8 weeks of age and older, hepatocellular carcinomas and hepatoblastomas in a background of liver dysplasia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Development of a transgenic mouse system for the analysis of stages in liver carcinogenesis using tissue-specific expression of SV40 large T-antigen controlled by regulatory elements of the human alpha-1-antitrypsin gene.

alpha-1-Antitrypsin (AAT) is the major antiprotease in human plasma; it is synthesized primarily in hepatocytes and to a lesser extent in several nonhepatic tissues. Under the control of regulatory elements of the human AAT gene, expression of SV40-large tumor antigen (T-ag) in transgenic mice occurred in the liver, stomach, pancreas, and kidney. Among seven founder transgenic animals, six developed liver carcinoma, four showed gastric neoplasia, and one developed pancreatic carcinoma. In three animals the kidneys showed glomerular or tubular epithelial hyperplasia but no malignancy. A stable transgenic line, 1812, was established. Members of this line reproducibly develop liver tumors by 10 weeks of age but do not exhibit any phenotypic changes in other tissues. Histological changes leading to liver tumor formation occurred with predictable kinetics and could be classified into four distinct stages: (a) embryonal/fetal stage, no recognizable histological changes; (b) newborn to 2 weeks of age, hyperplastic hepatocytes with reduced amounts of cytoplasm but no nuclear alterations; (c) between 3 and 8 weeks of age, diffuse liver cell dysplasia without observable tumor nodules; and (d) 8 weeks of age and thereafter, hepatocellular carcinomas in a background of liver dysplasia. Embryonic and newborn liver tissue showed uniform, high level expression of T-ag in the majority of hepatocytes by immunohistochemistry, whereas the dysplastic and tumoral stages were characterized by considerable variation in both the intensity of T-ag staining and the proportion of T-ag-positive cells. Immunoprecipitation analyses showed that T-ag was complexed with cellular protein p53 in all tumor samples. This study showed that SV40 T-ag expression in the liver resulted in cellular hyperplasia and dysplasia; additional event(s) apparently were required for progression to neoplasia. Those cooperating events occurred with predictable kinetics. This transgenic mouse system displays several similarities with human liver disease and provides a practical model for the study of separate steps in hepatocarcinogenesis.

Animals↗
PubMed1989

Identification of a conserved sequence in the non-coding regions of many human genes.

We have analyzed a sequence of approximately 70 base pairs (bp) that shows a high degree of similarity to sequences present in the non-coding regions of a number of human and other mammalian genes. The sequence was discovered in a fragment of human genomic DNA adjacent to an integrated hepatitis B virus genome in cells derived from human hepatocellular carcinoma tissue. When one of the viral flanking sequences was compared to nucleotide sequences in GenBank, more than thirty human genes were identified that contained a similar sequence in their non-coding regions. The sequence element was usually found once or twice in a gene, either in an intron or in the 5' or 3' flanking regions. It did not share any similarities with known short interspersed nucleotide elements (SINEs) or presently known gene regulatory elements. This element was highly conserved at the same position within the corresponding human and mouse genes for myoglobin and N-myc, indicating evolutionary conservation and possible functional importance. Preliminary DNase I footprinting data suggested that the element or its adjacent sequences may bind nuclear factors to generate specific DNase I hypersensitive sites. The size, structure, and evolutionary conservation of this sequence indicates that it is distinct from other types of short interspersed repetitive elements. It is possible that the element may have a cis-acting functional role in the genome.

Animals↗
PubMed1989

Spontaneous progression of hyperplastic outgrowths of the D1 lineage to mammary tumors: expression of mouse mammary tumor virus and cellular proto-oncogenes.

Mammary cancer in mice is characterized by progression through defined stages of preneoplasia, with the most common preneoplastic stage being the hyperplastic alveolar nodule (HAN). We determined the relative levels of RNA expression of various cellular proto-oncogenes and endogenous mouse mammary tumor virus genes in outgrowths and tumors of three sublines of the transplantable D1 HAN preneoplastic outgrowth line. The three sublines differed in relative tumor-producing capabilities. Subline D1B produced a high incidence of tumors with short latency periods, whereas sublines D1C and D1D produced low incidences of tumors with long latency periods. No consistent alteration in proto-oncogene expression correlated with relative tumorigenicity, although tumors frequently contained higher levels of one or more proto-oncogene transcripts as compared with preneoplastic tissue. Slightly elevated (2- to 6-fold) levels of different oncogene transcripts were detected in 13 of 17 tumors as compared with outgrowth tissue, including abl (2 tumors), fps (5 tumors), Ha-ras (6 tumors), and Ki-ras (8 tumors). One tumor contained 45 times more Ki-ras-specific RNA than outgrowth tissue because of a comparable amplification of Ki-ras DNA sequences. Elevated levels of Ha-ras occurred more frequently in tumors of a high-incidence subline than in a less-aggressive subline (5/10 vs 1/7), but this difference was not statistically significant. However, consistent changes in MMTV expression accompanied progression from preneoplastic tissues to mammary tumors. All 17 tumors displayed reduced levels of the MMTV-specific long terminal repeat (LTR) transcript (1.6 kb) as compared with HAN tissue; tumors with moderate levels of LTR transcript expressed the 3.8-kb envelope message as well, one not detected in HANs. Expression of the LTR transcript is apparently influenced by factors in addition to the methylation status of endogenous mouse mammary tumor virus genes, which was similar in outgrowths and tumors. As the survey of representative proto-oncogenes failed to identify a uniform change between HAN and tumors, it is likely that other genes are involved in tumor progression in the mammary gland.

Animals↗
PubMed1989

Solubilization of SV40 plasma-membrane-associated large tumor antigen using single-phase concentrations of 1-butanol.

The nature of the interaction of the simian virus 40 (SV40) transforming protein, large tumor antigen (T-ag), with the plasma membrane of transformed cells is not well understood. We report here that SV40 plasma-membrane-associated large tumor antigen (pmT-ag) can be solubilized by using single-phase concentrations of 1-butanol. Purified plasma membranes from SV40-transformed mouse cells yielded T-ag when treated with 2.5% butanol; solubilization of T-ag from the purified membranes in butanol was temperature dependent, with approximately 10-fold more T-ag extracted at 37 degrees C than at 22 degrees C; and application of 2.5% butanol to mKSA cells after cellular surface proteins had been radiolabeled with 125I resulted in the release of iodinated T-ag. Butanol-extracted pmT-ag coprecipitated with p53 and several cellular proteins ranging in size from 35 to 60 kDa. One cellular component migrated at a mobility similar to that of tubulin (56 kDa), and a monoclonal antibody against the alpha subunit of tubulin coprecipitated T-ag. Immunoblotting of proteins immunoprecipitated with monoclonal antibodies against T-ag or p53 from butanol extracts with a monoclonal antibody against the beta subunit of tubulin revealed specific coprecipitation of tubulin with T-ag and p53. This suggests that complexes composed of tubulin, T-ag, and p53 exist in butanol extracts. Control experiments eliminated the possibility of an artifactual association of tubulin with T-ag and p53 induced by butanol. Two-dimensional gel analyses revealed that 2.5% butanol at 37 degrees C extracted a subset of membrane-associated proteins and some cytosolic proteins, as well as a number of proteins that were not soluble in either high salt or detergent. Thus, the butanol extraction conditions employed in this study recovered a species of pmT-ag that appears to complex with tubulin. As butanol reportedly is less deleterious to native protein structures than other agents, including high salts and detergents, this extraction procedure may be useful for studying the structure and function of other membrane-associated proteins.

1-Butanol↗