Resolution of factors required for the initiation of transcription by yeast RNA polymerase II.

Fractionation of a yeast nuclear extract reveals at least four factors required in addition to RNA polymerase II for accurate initiation of transcription. One of these factors can be replaced by HeLa transcription factor IID or by its yeast counterpart expressed in Escherichia coli. Each of the remaining three factors can be replaced by a fraction from yeast whole cell extract, facilitating further purification of the factors.

Human papillomavirus type 18 in conjunctival intraepithelial neoplasia.

Human papillomaviruses are oncogenic viruses that have been found in a variety of epithelial neoplasias. We sought to confirm their presence in conjunctival intraepithelial neoplasia. Five tumors were studied with a polymerase chain-reaction assay designed to detect the E6 region of human papillomavirus types 16 and 18. Human papillomavirus type-16 DNA was found in four of the five tumors, including two tumors that contained both type-16 and type-18 DNA. Viral DNA was not present in the fifth tumor.

Autoantibodies from a patient with scleroderma CREST recognized kinetochores of the higher plant Haemanthus.

Human autoantibodies from a patient with scleroderma CREST (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, telangiectasia) were used to immunostain kinetochores on chromosomes in endosperm of the seed of the monocot Haemanthus katherinae Bak. Kinetochores of mitotic chromosomes and prekinetochores of interphase cells were specifically stained using conventional indirect immunofluorescence procedures as well as a nonfading immunogold-silver-enhanced technique and analyzed by fluorescence and video microscopy. In interphase, prekinetochores were either single or double structures depending on the stage of the cell cycle but became quadruple (two distinct stained dots on each chromatid) in mid-to-late prophase. In favorable preparations of prometaphase chromosomes, multiple subunits could be resolved within each sister kinetochore suggesting a compound organization. Western blot analysis demonstrated common epitopes in centromeric peptides of HeLa and Haemanthus cell extracts. Although the molecular mass of individual polypeptides differed in the two species, the presence of shared epitopes indicates striking conservation of centromere/kinetochore components throughout evolution.

Human immunodeficiency virus pseudotypes with expanded cellular and species tropism.

One mechanism for expanding the cellular tropism of a virus is through the formation of phenotypically mixed particles or pseudotypes, a process commonly occurring during viral assembly in cells infected with two or more viruses. We report here that dual infection of cells with human immunodeficiency virus (HIV) and a murine amphotropic retrovirus leads to the production of HIV pseudotypes that have acquired the host range of the amphotropic retrovirus and are capable of infecting not only CD4- human cells but also mouse cells. The replication of the HIV pseudotypes in the various CD4- cells was determined by measuring the appearance of HIV antigens in the supernatants, by cocultivation of CD4+ CEM cells with the infected CD4- cells, and in some cases by assaying the culture supernatants directly for infectious virus. Of the cells tested, human foreskin fibroblasts were the best host cells, and by in situ cytohybridization, we were able to document that all cells in the culture were infected. In addition, the temporal appearance of HIV-specific proteins in the HIV pseudotype-infected fibroblasts was similar to that seen in CD4+ CEM cells. If the human fibroblasts were first infected with the amphotropic retrovirus, they demonstrated the property of superinfection exclusion and were resistant to subsequent infection by the HIV pseudotype. In other cell lines, including the human glioblastoma-derived cell line U373MG, HeLa cells, BALB/c mouse embryo cells, and SC-1 wild mouse cells, although the HIV pseudotype infection appeared to be less efficient, substantial amounts of HIV were nevertheless produced. These results indicate that the HIV (amphotropic retrovirus) pseudotypes may be useful for studying the molecular biology of HIV infections in a wide range of cells.

Human Ro ribonucleoprotein particles: characterization of native structure and stable association with the La polypeptide.

Anti-Ro autoantibodies, found in sera of patients with systemic lupus erythematosus, Sjogren's syndrome, and related diseases, target the Ro ribonucleoprotein particles (RNPs). Although the polypeptide and RNA components of the Ro RNPs have been characterized, much less is known about the native structure of these particles. We have now characterized by biochemical techniques intact Ro ribonucleoprotein particles from cultured HeLa cells. These particles segregated in three discrete subpopulations with characteristic physicochemical properties: one containing hY5 RNA (RohY5 particles), one containing only hY4 RNA (RohY4 particles) and one with hY1, hY3, and hY4 RNAs (RohY1-hY4 particles). The RohY5 particles were purified free of contaminating ribonucleoproteins; both the La and the 60-kD Ro polypeptides were stable components of this portion of the Ro RNPs. The La RNPs co-purified with the RohY4 particles and contaminated the RohY1-hY4 RNPs. The stable association between the La and the 60-kD Ro polypeptides provides a potential macromolecular target for the linked set of anti-Ro and anti-La antibodies, and suggests a possible functional association of these polypeptides.

Bacteriophage T4 gene 32 protein shares antigen determinants with reconstituted HeLa hnRNP proteins in western blots.

A polyclonal antiserum against purified bacteriophage T4 gene 32 protein was raised in rabbits. In Western blots it detected a number of SDS-PAGE separated nuclear and ribosomal proteins of HeLa cells. Using a renaturing blotting system, however, larger hnRNP proteins ranging between 66.000 and 82.000 Da preferentially reacted with this antiserum. In addition hnRNP group A core proteins were detected to a minor extent. Nucleic acid binding proteins like histones or ribosomal proteins were not stained by this antibody after renaturation.

Towards microfluorometric quantitation of polyamines in situ. Relationship between cellular polyamine concentration and fluorescence yield of the formaldehyde fluorescamine method.

Two different fluorescence cytochemical methods, the formaldehyde-fluorescamine (FF) method and the orthophthalaldehyde (OPT) method as well as an immunocytochemical method have been developed for the localization of spermidine and spermine. Of these three methods, the FF-method is the most easy to perform. We have studied the relationship between fluorescence intensity induced by the FF-method and cellular polyamine levels measured by HPLC in MCF-7 cells and HeLa cells. The experiments were designed to obtain different cell concentrations of polyamines. Cells grown on microscope slides in Petri-dishes were partly depleted of spermidine by two days inhibition of their ornithine decarboxylase activity using alpha-difluoromethylornithine. One hr before harvest the cells were exposed to different concentrations (0-30 microM) of spermidine. Microfluorometric results and chemical determinations of spermidine and spermine were obtained from each separate slide. The cellular total polyamine (spermidine + spermine) concentration on the slides varied between 4 and 15 nmol per mg protein (MCF-7 cells) and 5 and 26 nmol per mg protein (HeLa cells) and the corresponding microfluorometric results between 60 and 115 arbitrary units (MCF-7 cells) and 80 and 160 arbitrary units (HeLa cells). Simple regression analysis showed a good linear relationship between cellular polyamine concentration and FF-fluorescence yield. The correlation coefficient for MCF-7 cells was 0.86 and for HeLa cells 0.82, significance of the correlations was p less than or equal to 0.0001. Our results add further credence to the specificity of the FF-method and indicate that the method may be useful for microfluorometric quantitation of polyamines in situ.

Effects of osmotic manipulation of intracellular hydration of HeLa S-3 cells on their proton NMR relaxation times.

Pellets of HeLa from suspension cultured cells in isotonic medium (300 mosmolar) were introduced into a Bruker CXP100 NMR spectrophotometer at 80 mHz within 5 min of the start of centrifugation. T1 and T2 times were measured within a total elapsed time of 20-25 min at 80 mHz and 37 degrees C, and averaged 1430 msec and 120 msec, respectively. Extrapolation to zero extracellular space gave a corrected T1 of 1370 msec. For cells collected after 10 min in hypotonic medium (down to 30 mosmolar) increased proton density correlated well with increased cell water content, but relaxation times did not rise in proportion to that predicted for the entry of "bulk" water (T1 of 4700 msec), except when swelling approached lysis point. Cells partially dehydrated by 10 min in hypertonic medium of up to 1500 mosmolar have also been analyzed, but once again the shortening of T1 was not proportional to the loss of "free" (bulk phase) water. At the upper limit of hypertonic treatment, lacunae or vacuoles of a watery nature separated within the cytomatrix, preventing maximum dehydration. The relationship of cell water to T1 is complex over the whole range of tonicity that HeLa S-3 cells tolerate. The data indicate, however, that hypotonically induced water probably has an average T1 time considerably lower than bulk phase water. In contrast, raising the total extracellular volume with medium had precisely the predicted effect on T1 time, further strengthening the case that water taken up by cell acquires a shorter T1 time. Cells adapting to hypotonic conditions oscillated in size and water content over 2-3 hr before returning to near their initial volume. Under these circumstances, T1 oscillated in the same way but with a reduced amplitude, consistent with the above findings.

Human lamin B contains a farnesylated cysteine residue.

We recently showed that HeLa cell lamin B is modified by a mevalonic acid derivative. Here we identified the modified amino acid, determined its mode of linkage to the mevalonic acid derivative, and established the derivative's structure. A cysteine residue is modified because experiments with lamin B that had been biosynthetically labeled with [3H]mevalonic acid or [35S]cysteine and then extensively digested with proteases yielded 3H- or 35S-labeled products that co-chromatographed in five successive systems. A thioether linkage rather than a thioester linkage is involved because the mevalonic acid derivative could be released from the 3H-labeled products in a pentane-extractable form by treatment with Raney nickel but not with methanolic KOH. The derivative is a farnesyl moiety because the Raney nickel-released material was identified as 2,6,10-trimethyl-2,6,10-dodecatriene by a combination of gas chromatography and mass spectrometry. The thioether-modified cysteine residue appears to be located near the carboxyl end of lamin B because treatment of 3H-labeled lamin B with cyanogen bromide yielded a single labeled polypeptide that mapped toward this end of the cDNA-inferred sequence of human lamin B.

The mammalian analogue of the yeast PRP8 splicing protein is present in the U4/5/6 small nuclear ribonucleoprotein particle and the spliceosome.

HeLa cell nuclear extracts contain a protein reactive with antibodies against PRP8, a polypeptide essential for pre-mRNA splicing in yeast and a specific component of the yeast U5 small nuclear ribonucleoprotein (snRNP) [Lossky, M., Anderson, G. J., Jackson, S. P. & Beggs, J. (1987) Cell 51, 1019-1026]. The mammalian protein appears as a doublet at approximately 200 kDa, smaller than the 260-kDa yeast protein, and possesses an Sm epitope as determined by immunoblotting. Its association with a snRNP of the Sm class other than U1 or U2 is indicated by its immunoprecipitation by anti-Sm and anti-trimethylguanosine antibodies but not by anti-(U1) or anti-(U2) RNP sera. Gradient fractionation of splicing extracts demonstrates that the 200-kDa protein is a component of the U4/5/6 snRNP complex and of U5 snRNPs. It is also present in affinity-purified spliceosomes.

Detection of human papillomavirus DNA in cervical cancer tissue by the polymerase chain reaction.

A method for detecting HPV DNA in cervical cancer tissue was developed without using isotopes. The DNA samples from the cancer tissues were first subjected to amplification by PCR, followed by polyacrylamide gel electrophoresis to identify the specific amplified fragment. The specificity and sensitivity of the PCR method are described. Compared with the dot hybridization technique, it is shown that the method is able to detect HPV DNA in cervical cancer tissues.

Rapid isolation of DNA from complex biological samples using a novel capture reagent--methidium-spermine-sepharose.

We have synthesized and analyzed the functional properties of a novel DNA capture reagent containing a methidium moiety attached to a sepharose bead by a spermine linker. DNA present in a biological fluid or other complex sample binds to the reagent. The DNA-capture reagent complex is then separated from the sample by centrifugation and the DNA is released from the reagent by brief incubation in 0.1 to 0.5 N NaOH or KOH. Capture of DNA from complex samples is independent of the salt concentration of the sample, and occurs in the presence of high concentrations of EDTA, proteinase K and detergents. Many samples can be processed simultaneously. The following specific applications, in which denatured DNA is quantitated or characterized, are demonstrated: 1). Isolation of hepatitis B virus DNA from serum and quantitation by dot-blot hybridization, 2). Isolation and quantitation of DNA from urine, 3). Isolation of human genomic DNA from one microliter of blood or 100 HeLa cells followed by amplification of a specific gene sequence using the Polymerase Chain Reaction, 4). Isolation of single stranded phage M13 sequencing templates from bacterial cultures. These investigations suggest that a capture reagent containing an intercalating moiety bound to a solid support may be useful for many applications in molecular biology and molecular diagnostics.

20S small nuclear ribonucleoprotein U5 shows a surprisingly complex protein composition.

U5 small nuclear ribonucleoprotein (snRNP), purified from HeLa nuclear extracts (splicing extracts), shows a complex protein composition. In addition to the snRNP proteins B', B, D, D', E, F, and G, which are present in each of the major snRNPs U1, U2, U4/U6, and U5, U5 snRNP contains a number of unique proteins characterized by apparent molecular masses of 40, 52, 100, 102, 116, and 200 (mostly a double band) kDa. The latter set of proteins may be regarded as U5-specific for the following reasons. They are not only eluted specifically, together with snRNP particles, from anti-2,2,7-trimethylguanosine immunoaffinity columns by 7-methylguanosine, they also cofractionate with U5 snRNP during chromatography and, most importantly, in glycerol gradient centrifugation. These U5 snRNP particles show a high sedimentation constant of about 20S. U5 snRNPs that lack the U5-specific proteins are also found in nuclear extracts but have (in comparison) a lower sedimentation value of only 8-10S. Autoimmune sera from patients with systemic lupus erythematosus were identified that, on immunoblots with purified U5 snRNP proteins, reacted selectively with the 100- or 200-kDa proteins. This indicates that at least the high molecular mass U5-specific proteins are structurally distinct and not derived one from the other by proteolytic degradation. The existence of so many unique proteins in the U5 snRNP suggests that this snRNP particle may exert its function during splicing mainly by virtue of its protein components.

Human nuclear protein interacting with a conservative sequence motif of Alu-family DNA repeats.

Human retrotransposons, Alu-family DNA repeats (AFRs), have variable nucleotide sequence but conservative short elements, which may have important functions, are also present. In our previous reports we have described human nuclear DNA-binding protein interacting with AFRs and evidence was presented that the protein recognizes sequence motif 5'-GGAGGC-3' which is conserved in the spacer of RNA polymerase III promoter of AFRs and in the SV40 T-antigen-dependent replication origin of AFRs. In this study it was found that double-stranded synthetic oligonucleotides containing indicated conservative sequences of AFRs actually have high-affinity binding site for HeLa nuclear protein. The data suggest that non-infected human cells contain nuclear DNA-binding protein which recognizes the conservative sequence motif of AFRs - GGAGGC.