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Efficient transfection of eukaryotic cells is dependent on the purity of the transfected genomic DNA. In an attempt to obtain a more reliable method of DNA purification we have modified the widely used protocol of Blin and Stafford to include a treatment with guanidinium thiocyanate. The DNA obtained following the present protocol transfects eukaryotic cells with higher efficiency.

Plasmacytoma M603 contains one normal, nontranslocated c-myc gene and one translocated c-myc gene in which c-myc exon 1 is juxtaposed with the immunoglobulin heavy chain enhancer and c-myc exons 2 and 3 are juxtaposed with C alpha. We find that steady-state c-myc RNA levels are 2-4 fold elevated in M603 relative to normal liver or spleen and that these transcripts originate predominantly if not exclusively from the translocated c-myc gene. Although both promoters on the nontranslocated c-myc gene are repressed, the proximal promoter, P1, is active on the translocated 5' c-myc region which is juxtaposed with the immunoglobulin heavy chain enhancer. The 3' portion of the translocated c-myc gene is transcribed from fifteen cryptic start sites and spliced at aberrant donor and acceptor splice sites, thereby generating a mixture of transcripts with different, abnormal 5' untranslated regions. Although the reason that translocation activates the cryptic c-myc starts in M603 is not completely understood, we show that truncation of the c-myc gene is not sufficient to activate cryptic transcription sites.

Redistribution of concanavalin A receptors on the surface of normal and transformed cell lines was studied by indirect immunofluorescence. Normal murine fibroblasts are compared with line L transformed murine cells, whereas NRK line cells from normal rat kidney with their transformed counterparts of Ki-MSV line. It was shown that the total area occupied by the transformed cells on the substratum and the size of lamellar cytoplasm (i. e. the peripheral part attached to the substratum) are diminished as compared to those in normal cells. The surface zone from which concanavalin A-agglutinated receptors are eliminated is approximately equal to the area of lamellar cytoplasm of normal cells and is less as compared to that of transformed cells.

Myosin has been purified from the following cultured cell lines: normal rat kidney fibroblast (NRK), HeLa-Rhino (HeLa), human choriocarcinoma, human acute lymphoblastic leukemia, rat hepatoma (HTC), monkey kidney (VERO), pigmented mouse melanoma, Y-1 rat adrenal cortex, and growth hormone-secreting GH-1. Myosin constitutes 0.5-5.4% of the protein of these cells. It was not detected in washed human erythrocytes or in two types of mouse plasmacytoma cells. Two methods for the purification of myosin from cultured cells have been employed. With Method I highly purified myosin was prepared by Sepharose 4B and DEAE-cellulose chromatography from 10(10) L-929 cells as well as from mouse uterus. Those myosins have similar molecular and subunit weights as well as ATPase activity but are immunologically distinct. Method II involving ultracentrifugation and Sepharose 4B chromatography, is suitable for the production of moderately pure myosin in good yield from as few as 5-10(7) cells (five 100-mm Petrie dishes).

Fluorescence polarization of cis- and trans-parinaric acid and high angle x-ray diffraction are used to examine the physical effects of in vitro sterol depletion of LM cell plasma membranes. Measurements on lipids isolated from the normal plasma membrane show that most of the lipid is fluid between 5 and 45 degrees C. When no sterol is present, approximately 15% of the phospholipids undergo gel phase formation, detectable by x-ray measurements, in this temperature range. Partial sterol repletion of the isolated phospholipids serves to lower the onset temperature of the phase transition, as well as to decrease the fraction of lipid undergoing a transition. Intact plasma membranes are sterol depleted by in vitro incubation with egg phosphatidylcholine liposomes. This induces a phase transition with an onset temperature increasingly above the physiological temperature as more sterol is removed from the membranes. The transition can be suppressed by returning sterol to the membranes. These experiments extend earlier work (Rintoul, D. A., Chou, S.-M., and Silbert, D. F. (1979) J. Biol. Chem. 254, 10070-10077) on isolated plasma membrane lipids by showing that sterol suppresses a phase transition in intact membranes as well as in isolated lipids. The correlation between gel phase formation upon sterol loss at the physiological temperature and loss of membrane glucose transport function is discussed.

Using DNA from L cells which expressed high levels of the CD7 (Leu-9 or HuLy-m2) antigen obtained after two cycles of transfection, a genomic library was constructed in the lambda phage Charon 4A. Recombinant clones containing the gene coding for this antigen were identified by first screening the library with both the HSV-tk gene and a probe detecting the human repetitive (Alu) sequences. DNA from 10 tk+ and 12 Alu+ recombinant clones was used to transfect L cells which were analyzed for the cell-surface expression of CD7 either early (48-72 h posttransfection) or later when hypoxanthine aminopterin thymidine-resistant colonies were obtained. Transfection with either Alu+ or tk+ recombinant phages led to transient early expression of CD7, and stable CD7+ transfectants were also established. Thus the CD7 gene has been isolated in a number of clones in association with either the Alu repetitive sequence or with the HSV-tk gene; the insert size in one of the genomic clones was 13.5 kb.

The base sequence of mouse L-cell satellite DNA was investigated by degradation of the two separated complementary strands with the base specific enzyme, T4 endonuclease IV. Digestion of the heavy strand DNA released a limited number of oligonucleotides which were separated by ionophoresis/homochromatography, isolated, and sequenced by the 'wandering spot' method. The light strand DNA was resistant to digestion with T4 endonuclease IV and no detectable amounts of oligonucleotides were released. The oligonucleotides obtained from the heavy strand were related in sequence, indicating that mouse satellite DNA derived from a short tandemly repeated sequence. The sequence of part of the original repeat unit is proposed to be C-A-T-T-T-T-T-C. Five major oligonucleotides were identified, all of which differ from the proposed original sequence by single base changes. The five major oligonucleotides occur with about equal frequency and together comprise approximately 50% of the oligonucleotides released by T4 endonuclease IV from the heavy strand DNA. In addition to the five major oligonucleotides, several oligonucleotides were found to occur in lesser amounts. Since these oligonucleotides are related to the major oligonucleotides, it is likely that they have arisen from them by mutation.

The polar lipid patterns of several cell strains (EUE, L929, MT, RAG and TAZA) were compared by TLC separation in order to identify a typical lipid profile as a possible "strain marker". Phospholipids and sulfolipids were tested by colorimetric procedures. EUE and L929 contain smaller amounts of sulfolipids (2.4-3.9; 3.0-4.5) than RAG (4.4-12.0) or MT (10.5) (micrograms x 10(-6)/cell). MT and RAG contain higher phospholipid P concentration than other strains. (MT:14.0 whilst EUE 1.3-1.8 micrograms phospholipidic P x 10(-6)/cell). The patterns of lipidograms in the characterization of two cell strains are discussed in terms of their usefulness as rapid "fingerprint" in biochemical and histochemical analysis.

The sequence content of mouse L cell pre-rRNA was examined by RNA gel transfer and blot hybridization. Nuclear RNAs were separated by agarose gel electrophoresis, transferred to diazo-paper, and hybridized to twelve different restriction fragments that are complementary to various sections of 45S pre-rRNA. An abundant new 34S pre-rRNA and less abundant new 37S, 26S and 17S pre-rRNAs were detected. The presence of these new pre-rRNAs suggests the existence of at least two new pre-rRNA cleavage pathways. 34S and 26S pre-rRNAs were also detected in HeLa cells suggesting that these new cleavage pathways are characteristic of mammalian cells. Further, an abundant new 12S precursor to 5.8S rRNA was also detected and is common to all the proposed cleavage pathways. The previously identified 45S, 41S, 32S and 20S pre-rRNAs were readily detected and their general structure confirmed. The 20S pre-rRNA is characteristic of the known pathway used by HeLa and other cells, and its presence suggests that growing mouse L cells use this pre-rRNA cleavage pathway. The 36S pre-rRNA characteristic of the previously described mouse L cell cleavage pathway was not detected. In all these cleavage pathways pre-rRNA cleavage sites are apparently identical and occur at or near the termini of the mature 18S, 5.8S and 28S rRNA sequences. The pathways differ only in the temporal order of cleavage at these sites. The position of the 5.8S rRNA sequence was located within the internal transcribed spacer. The known and conserved sequence of 5.8S rRNA from several organisms predicts a characteristic pattern of restriction enzyme sites for 5.8S rDNA. Internal transcribed spacer rDNA was mapped with restriction enzymes, and the characteristic pattern was found near the midpoint of the internal transcribed spacer. This places the 5.8S rRNA sequence at or near the 5' terminus of 32S pre-rRNA.

Rat lymphotoxin (LT), a soluble mediator which kills L cells (mouse fibroblasts) within 48 hr, was partially purified by conventional protein fractionation procedures from the culture supernatants of rat lymph node cells stimulated with ovalbumin in vivo and rechallenged with the same antigen in vitro. It appeared, from the elution pattern of DEAE-cellulose chromatography, to be a neutral or slightly basic protein and was inactivated at 60 degrees C. Its molecular weight was estimated by Sephadex gel filtration to be approximately 9 times 10(4) daltons. Partially purified LT, in titration studies, showed single hit kinetics. It also inhibited L cell proliferation at low concentration without significant killing effect. The heat inactivation curves of both cytotoxic and proliferation inhibitory activities were the same, suggesting that these two activities may be caused by the same molecule. However, it had almost no detectable effect on the DNA synthesis of rat lymph node cells stimulated with phytohemagglutinin (PHA), suggesting that lymphocytes, at least those which respond to HA, are rather resistant to LT. It is suggested that activities described as "proliferation inhibitory factor" and "cloning inhibitory factor" by others may represent the activity of dilute LT. The "inhibitor of DNA synthesis" is distinct in molecular character, range of target cells, and target cell kinetics from LT.

About 2% of mouse DNA reassociates at extremely low Cot values (10-7-10-6 mole-liter-1-sec). This DNA fraction was isolated with the aid of nuclease S1 and purified by chromatography on hydroxyapatite. The study of this fraction suggests that it has a structure of hairpin type. The DNA of the hairpins can hybridize with sequences forming the double-stranded regions in pre-mRNA. Probably at least some of the DNA of the hairpins, described as "reversed repeating sequences" of DNA, is transcribed with the formation of structures of hairpin type in pre-mRNA.

The kinetics of hybridization of polyadenylated RNA from mouse L-cells with complementary DNA (cDNA) synthesized with reverse transcriptase revealed three classes of differing abundance. The simplest interpretation requires three frequency classes representing polyadenylated RNA; 5, 45, and 50 percent of the total polyadenylated RNA and about 3, 300, and 7600 different RNA sequences of 6 times 10-5 daltons, respectively. The complementary DNA synthesized with L-cell polyadenylated RNA as template hybridized efficiently with RNA from different mouse tissues, indicating that most species of the L-cell RNA in the highand middle frequency class are present in all mouse tissues. Kinetics of hybridization of complementary DNA synthesized with cytoplasmic polyadenylated brain RNA as template suggested a higher complexity for brain RNA. Thirty-five percent of this brain cDNA failed to hybridize with L-cell RNA. This complementary DNA fraction, isolated by hydroxylapatite chromatography, represented approximately 11,000 RNA sequences specific for the brain. On the other hand, hybridization of complementary DNA synthesized on polyadenylated mouse liver RNA with L-cell RNA failed to demonstrate differences between these two groups of polyadenylated RNA.

The 5-methylcytosine residues of L-cells have been labeled with [methyl-3H]-L-methionine and their chromatin localization studied using deoxyribonucleases. The kinetics of micrococcal nuclease digestion showed that the methylated cytosine residues are concentrated within regions resistant to nuclease digestion and preferentially missing from those regions between nucleosomes which are nuclease sensitive. Using DNA hybridization kinetic analysis, it is shown that 5-methylcytosine is abundant in highly repeated sequences but is also present in middle repetitive and unique sequence DNA.

When chromosomal proteins in chromatin or in mononucleosomes were extensively cross-linked with an imido ester, the H1-containing nonameric histone complex was revealed. In this complex, histone H1 is connected with the octamer of core histones. The cross-linking of H1 of the octamer is realized preferentially through H2a and H3 histones. Some HMG (high mortality group) proteins located presumably in the linker regions of a nucleosome fiber also take part in the formation of dimers, possibly with the histones of a nucleosomal core. The results suggest mutant interactions between some linker-associated proteins and intranucleosomal histones. Experiments involving extensive cross-linking of proteins in the purified mononucleosome subfractions demonstrated differences in the organization of core histones between 'complete' nucleosomes and nucleosomes lacking H1.

Coxiella burnetii established a persistent infection of various cell lines including L929 mouse fibroblasts. Although the basis for such persistence is unknown, the phenomenon does require continual growth, proliferation, and maintenance of viability of the host cells. We examined the effect of short- and long-term infection on the host cell's generation time and cell cycle. Flow cytometric studies of actively growing normal and infected cells stained with mithramycin or propidium iodide revealed no significant difference in cell cycle distribution patterns or changes in ploidy level associated with persistent infection with either phase I or phase II C. burnetii. The population doubling times of infected and normal cells were similar.

Recently, we reported a multicopy mammalian plasmid with a structure related to polyoma. The plasmid, named L factor, was found at a high copy number (5,000 or more per cell) in a subclone derived from mouse L cells. We attempted to utilize L factor as a plasmid vector for mammalian cells. A series of composite DNA consisting of L factor and a foreign (herpes simplex virus tk) were constructed. These DNA could be established as plasmids after transfection to several mouse cell lines, although the copy number of the re-established plasmids was considerably less than that observed for the original subclone. The composite DNA maintained the structure of the original DNA after prolonged culture and the copy number remained constant even with no selective pressure. A composite DNA, with no DNA sequence corresponding to polyoma T antigen, could also be established as a plasmid in a mouse L cell line in which polyoma T antigen is expressed. The potential use of the plasmid is discussed.

A specific class of DNA sequences, the inverted repetitive sequences, forms a double-stranded structure within a single linear polynucleotide chain in denatured DNA. The reassociation process is unimolecular and occurs very fast. Quantitative analyses have shown that these sequences comprise about 4--5% of the nuclear DNA of various mammalian cells (P815 mouse mastocytoma, Hela, L cells, Raji and Chang cells, and human embryonic hepatocytes) and are interspersed within sequences of other degrees of repetitiveness. After labeling the cells with L-[Methyl-3H]methionine and [14C]deoxycytidine, relative rates of enzymic DNA methylation were computed on the basis of 3H and 14C radioactivities found in pyrimidine residues of the nuclear DNA. The results indicate that DNA of inverted repetitive sequences is methylated to a level about 50% higher than the ordinary repetitive sequences and to about 300% higher than the unique and intermediary sequences. The biological function of the inverted repeats as well as the role of their enzymic hypermethylation is unknown.

Mitotic chromosomes of L cells (metaphase plates) were dehistonized by centrifugation through a layer of 2 M NaCl and then treated with restriction endonuclease Bam HI. Alternatively, they were pretreated with EcoRI endonuclease, dehistonized, and additionally digested with EcoRI or HindIII. The DNA remaining attached to the axial structure of the chromosomes was isolated and investigated in renaturation experiments. It was found to be enriched in reiterated base sequences belonging to the satellite and to abundant intermediate repeats. The CsCl density gradient ultracentrifugation of this DNA separated the satellite from the fraction containing intermediate repeats.

Although the natural host of polyoma virus is the mouse, its integration in cellular DNA has been investigated almost exclusively in rat cells. We report here studies on the integration of polyoma in mouse cells. We introduced the polyoma virus genome in two different mouse cell lines as an unselected genetic marker, by cotransfection with the tk gene of herpes simplex virus or the neo gene of E. coli. The number of TK+ or G418R clones obtained was reduced up to 50 fold by the presence of the polyoma genome. The gene coding for the early protein large T of polyoma was necessary and sufficient to produce this reduction. However, this effect appeared to be independent of polyoma replication. Surprisingly, all of the 33 clones analysed that had survived cotransfection with polyoma contained polyoma DNA integrated in their genome. Furthermore, in over 50% of these clones, the entire polyoma genome had been integrated. We conclude that polyoma integrates readily in mouse cellular DNA.

Mature, closed circular mouse mitochondrial DNA contains a significant number of ribonucleotides throughout the genome. Previous studies have implicated the two origins of DNA replication as preferred sites of ribonucleotide retention. We have analyzed the site specificity of ribosubstitution by direct sizing of alkali-treated restriction fragments in comparison with the DNA sequence of untreated restriction fragments of cloned mouse mitochondrial DNA. These results have confirmed the observations that ribonucleotides are retained at the two origins of replication and are most likely remnants of RNA priming events associated with DNA replication. The map location of ribonucleotides at the light strand origin of replication has been refined to a triplet nucleotide (5'-CGG-3') in the light strand initiation region. This approach has demonstrated that all four deoxyribonucleotides are subject to ribosubstitution and no single base (or subset of the four bases) predominates. An examination of selected regions of the mitochondrial DNA genome including the putative coding region for cytochrome oxidase subunit III and regions containing the genes for tRNAPhe, tRNAVal, 12 S rRNA, and 16 S rRNA reveals preferred sites for ribosubstitution. These preferred sites do not relate in any obvious way to the functional aspects of these domains. In addition, the data indicate that every position in the DNA sequences examined can be ribosubstituted at a very low frequency.

The pseudouridine contents of the rRNA species of HeLa cells, mouse L-cells and Xenopus laevis cultured kidney cells were examined. Pseudouridine, like 2'-O-methylation, was found to occur relatively frequently in each of the high-molecular-weight rRNA species. However, the numerical data do not support the idea that there is a general one-to-one relationship between pseudoridine residues and 2'-O-methyl groups in vertebrate rRNA.

1. Sodium dodecylsulfate--polyacrylamide gel electrophoresis has been employed to analyze the protein and glycorprotein components of phagosome membranes prepared from mouse L cells by the polystyrene latex bead method. 2. These experiments showed that phagosome membranes contain at least 20 major membrane protein species having apparent molecular weights between 27,000 and 250,000; the most abundant proteins have molecular weights of 112,000, 103,000, 76,000, 68,000, 62,000, 42,000 and 36,000. 3. Phagosome membrane glycoproteins, which were detected on the gels by staining with periodic acid-Schiff's reagent, were found to migrate in two broad zones centered at positions on the gel corresponding to proteins of mol. wt 140,000 and 85,000. 4. Comparison of the phagosome membrane results with the results of similar experiments carried out with cell surface membranes revealed a high degree of similarity between the two. All major protein and glycoprotein components present in phagosome membranes could be identified in both of two types of cell surface membrane preparations analyzed. 5. These results strongly support the view that phagosome membranes contain a representative, not a highly selected, sample of surface membrane proteins and glycoproteins.

The Chinese hamster thymidine kinase (TK) gene has been isolated from a recombinant phage library constructed with genomic DNA from mouse Ltk- cells transformed to Tk+ by transfection with Chinese hamster genomic DNA. The phage library was screened by the Benton-Davis plaque hybridization technique, using as probes, subclones of recombinant phage that were isolated from mouse Ltk+ transformants by the tRNA suppressor rescue method. The Chinese hamster TK gene is contained within 13.2 kilobases of genomic DNA in the isolate designated lambda 34S4. This gene, defined by restriction enzyme sensitivity experiments, homology studies with the chicken TK gene, and mRNA blotting experiments, may extend over 8.5 kilobases. Subclones of the lambda 34S4 isolate used as hybridization probes identified a 1,400-nucleotide polyadenylated RNA as the hamster TK mRNA. The abundance of this mRNA varies dramatically in Chinese hamster cells cultured under various growth conditions, providing direct evidence that the growth dependence of TK activity may be regulated in an important way at the level of cytoplasmic TK mRNA.

The cytochemical properties of a guanine-specific synthetic fluorescent analogue of actinomycin D, 7-amino-actinomycin D, have been studied in fixed and living preparations of L cells and polytene chromosomes of salivary glands of Chironomus thummi thummi and Drosophila lummei (Hackman). 7-Amino-actinomycin D has been shown to bind to DNA-containing structures, thereby inducing in them a bright red fluorescence. No specific fluorescence has been found in RNA-containing structures treated with this fluorescent probe. The fluorescence pattern of some regions of polytene chromosomes with a known nucleotide composition was analysed. It has been established that 7-amino-actinomycin D induces a very weak fluorescence in GC-poor chromosome regions of the Drosophila lummei toromere structure. Data indicating a nonlinear dependence between the fluorescence intensity of a stained chromosome region and the GC content in its DNA have been obtained. The influence of DNA nucleotide composition in a chromosome region on the fluorescence of 7-amino-actinomycin D is discussed. In combination with quinacrine staining and the Feulgen fluorescence reaction, treatment with 7-amino-actinomycin D provides useful information about the distribution of GC base pairs in the chromosome region under study.

Squalene and sterol carrier protein (SCP) levels and sterol/phospholipid molar ratios of whole cells and plasma membranes were measured in cultured primary tumor and metastatic cell lines. SCP is abundant in all cell lines. However, metastatic lines have significantly lower SCP levels and plasma membrane sterol/phospholipid ratios than do primary lines. The results indicate that extremely malignant, metastatic cells are unable to produce or maintain adequate levels of both SCP and plasma membrane sterols when grown in lipoprotein deficient media. This defect, in vivo, probably causes excess uptake of SCP and lipid.

A factor inducing differentiation of mouse myeloid leukemic cells (MI) into macrophages was purified to apparent homogeneity from 168 1 of CM of Ehrlich ascites tumor cells. The purified factor was half-maximally active at 2 X 10(-11) M. The factor was analyzed by radioiodination, SDS-polyacrylamide gel electrophoresis and autoradiography. Its Mr was 40 000-50 000. On reduction, the factor lost activity, but showed no subunit structure. Treatment of the factor with endo-beta-N-acetylglucosaminidase F, but not endo-beta-N-acetylglucosaminidase H, gave rise to a molecule of Mr 20 000-28 000. The activity of the factor from Ehrlich cells was completely neutralized by antiserum to the factor of Mr 50 000-70 000 from mouse fibroblast L929 cells.

The firefly enzyme luciferase catalyzes the luminescent reaction of luciferin with ATP and oxygen. The luciferase gene has recently been cloned and proposed as a reporter gene in procaryotic and eucaryotic cells. We present here a luciferase activity assay which relies on luminescence detection using a standard scintillation counter. This technique is simple, fast, inexpensive, and still very sensitive: as little as 0.02 pg (250,000 molecules) of enzyme is readily detected. The technique is optimized for the luciferase assay in mammalian cell lysates. Thus, the luciferase gene may become a very useful tool for gene regulation studies.

The possibility of specific detection of interferon antigens by ELISA was demonstrated on a model of partially purified and ultrafiltration-concentrated preparations of mouse fibroblast interferon. ELISA allows differentiation of specific interferon antigens and the main protein contaminants of its preparations: bovine serum albumin and virus-inducer proteins.

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane.

We have isolated and sequenced a cDNA clone encoding the human lymphocyte receptor for IgE (Fc epsilon R). The deduced protein sequence reveals that Fc epsilon R consists of 321 amino acids, without any signal sequence, and is oriented with its N-terminus on the cytoplasmic side and its C-terminus on the outside of the cell. This molecule shows striking sequence homology with chicken asialoglycoprotein receptor (hepatic lectin), suggesting a possible role for Fc epsilon R in endocytosis. Fc epsilon R mRNA is expressed in B cells, B cell lines, and macrophage cell lines. It is not expressed in T cells or T cell lines, with the exception of an HTLV-transformed T cell line. mRNAs expressed in a macrophage line and in the latter T cell line differ in size from mRNA expressed in B cells. Human BSF-1 (or IL-4) induces the expression of Fc epsilon R mRNA in B cells, but not in T cells.

The band of cell fluorescence with the maximum of 395-400 nm is registered. This band is exposed on the phone of the tryptophane by wavelength excitation lambdaex=250-260 nm, and in pure scape by lambdaex=310-326 nm. Pyridoxin - substrate of vitamin B6 has identical parameters of spectra excitation and emission of neutral (pH 7) and acid (pH 2) solutions. After temperature damage of cells the intensity of this band increases.

When intact nucleoli were prepared in the presence of enough leupeptin and phenylmethanesulfonyl fluoride to inhibit protease action, electrophoretic patterns of their constituent proteins were reproducible and very similar for L, HeLa, CHO, and rat hepatoma cells. "Core nucleoli", defined as that nucleolar fraction which remains after extensive DNase I action, had a protein composition similar to that of crude intact nucleoli, but were enriched for snRNA U3. Core nucleolar proteins included all of the histones, ribosomal proteins, and phosphorylated proteins with mobilities corresponding to 110 (protein C23) and 160 kilodaltons (kDa). The presence of protein C23 and of lamins A and C in nucleoli and core nucleoli was further verified by reaction with specific antibodies after one- or two-dimensional electrophoresis. A class of higher molecular weight proteins, ranging from 70 to greater than 200 kDa by mobility, was observed. It included at least 25 specific proteins, almost all of them highly acidic (pI less than 3.5). Treatment of core nucleoli with ethylenediaminetetraacetic acid/hypotonic buffer solubilized 30-35% of the small and large molecular weight proteins. In contrast, washing core nucleoli with 2 M NaCl selectively released U3 snRNA, 95% of the ribosomal RNA, and about half of the proteins, including C23 and most of the histones, ribosomal proteins, and other lower molecular weight proteins. The fraction remaining insoluble, "nucleolar matrix", was enriched for proteins of 34 and 57 kDa, lamins A and C, and most higher molecular weight proteins, as well as a portion of ribosomal spacer DNA.