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H Weintraub

Publications and source records attributed to H Weintraub.

At least 109 records · Page 6Linked to original sources
PubMed1986

Expression of transfected DNA depends on DNA topology.

Supercoiled DNAs, especially those containing enhancers, yield markedly higher levels of expression than linearized DNA when transfected into CV-1 cells or L cells. Different templates, linear or supercoiled, enhancer-containing or not, saturate for expression at 2 micrograms DNA per dish, suggesting that one role for enhancers and supercoiling is to increase the efficiency with which the same limiting component is used. Plasmids containing only enhancers or only promoters do not compete for expression with an enhancer-driven gene. However, plasmids that contain both enhancers and promoters do complete, suggesting that a second role for enhancers is to increase the binding of a limiting transcription factor. Linear and supercoiled enhancer-promoter plasmids compete equivalently. This suggests that supercoiling affects the ability of transcription factors to activate a given promoter, once bound.

Acetyltransferases↗
PubMed1985

Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA.

Plasmid DNA directing transcription of the noncoding (anti-sense) DNA strand can specifically inhibit the expression of several test genes as well as normal, endogenous genes. The anti-sense plasmid constructions can be introduced into eukaryotic cells by transfection or microinjection and function in both transient and stable transformation assays. Anti-sense transcripts complementary to as little as 52 bases of 5' untranslated target gene mRNA specifically suppress gene activity as well as, or more efficiently than, anti-sense transcripts directed against the protein coding domain alone. Conditional anti-sense inhibition is accomplished with the use of hormone-inducible promoter sequences. Suppression of endogenous actin gene activity by anti-sense RNA is detected as a decrease in growth rate and as a reduction in the number of actin microfilament cables. These observations suggest that anti-sense RNA may be generally useful for suppressing the expression of specific genes in vivo and may be a potential molecular alternative to classical genetic analysis.

Acetyltransferases↗
PubMed1985

Ligand-blotting visualization of the LDL receptor in plasma membranes and in two classes of coated vesicles from adrenocortical cells.

Two populations of coated vesicles, different in size, have been isolated from the bovine adrenal cortex. The enrichment of the LDL receptor from the plasma membrane to the large coated vesicles and then to the small ones was evidenced by ligand-blotting ELISA assays. The LDL receptor has been characterized as a 130-kDa proteic component which retains the binding specificity and structural features in plasma membranes as well as in the two classes of coated vesicles.

Adrenal Cortex↗
PubMed1985

Receptor-mediated endocytosis of human transferrin and its cell surface receptor.

We have studied the process of transferrin endocytosis in human erythromyeloid cell line K562 using fluorescein (FL) and rhodamine (RD) labeled iron-saturated transferrin (FeTF), and a fluorescein labeled monoclonal antibody to the transferrin receptor (FL-mAB). Because the antireceptor antibody and FeTF bind to different sites on the TF receptor molecule, it is possible to simultaneously and independently follow receptor and ligand. We have measured the relative amounts of transferrin or antireceptor antibody bound in the presence or absence of proteolytic enzymes using a cell sorter. At 4 degrees C almost all of the FL-TF and the FL-mAB is surface bound in a diffuse pattern. Within minutes of elevating the temperature to 37 degrees C surface aggregates form and the FL-TF is internalized. At this time about one sixth of the transferrin is still surface bound and accessible to papain digestion. The remainder localizes in a perinuclear cluster of vesicles. Monoclonal antibody binds to the cell surface transferrin receptor but is not internalized at 4 degrees C or 37 degrees C. When unlabeled diferric transferrin is added, it promotes the uptake of the Fl-mAB. The addition of goat anti-mouse immunoglobulin also promotes FL-mAB uptake. These studies support the concept that a specific trigger is required for transferrin receptor endocytosis.

Animals↗
PubMed1985

Localization of DNAase I-sensitive sequences to specific regions of interphase nuclei.

Active or potentially active gene loci are preferentially sensitive to DNAase I digestion. Cedar and colleagues used this DNAase I sensitivity to label active sequences preferentially by nick translation of nuclei and chromosomes. Using biotinylated nucleotides and immunofluorescence detection, we have looked for compartmentalization of DNAase I-sensitive sequences by nick translation of nuclei maintained as three-dimensional structures. Labeled sequences in mouse L cells are preferentially localized at the nuclear periphery in both permeabilized nuclei and frozen sections. In newt and chicken nucleated erythrocytes, labeled regions are at borders of condensed chromatin masses along interchromatin channels communicating with the nuclear periphery. Control experiments indicate that nick translation of nuclei preferentially labels highly expressed genes and that the preferential localization of DNAase-I sensitive regions is probably not a consequence of fixation artifacts or selective nuclear permeability to the labeling reagents.

Animals↗
PubMed1985

Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA.

The bacteriophage SP6 promoter and RNA polymerase were used to synthesize sense and antisense RNAs coding for the enzymes thymidine kinase (TK) and chloramphenicol acetyl transferase (CAT). Injection of antisense CAT RNA into frog oocytes inhibited expression of sense CAT mRNA. Similarly, antisense TK RNA inhibited expression of sense TK mRNA. Antisense RNAs were stable in oocytes and had no detectable effect on either the expression of endogenous proteins or on the expression of nonhomologous RNA transcripts. CAT activity expressed from a plasmid transcribed in the oocyte nucleus was also inhibited by antisense RNA injected into the oocyte cytoplasm. The data suggest that antisense RNA will be useful in identifying the function of specific mRNA sequences during early development of the frog.

Acetyltransferases↗
PubMed1985

Neurological mutation characterized by dysmyelination in NCTR-Balb/C mouse with lysosomal lipid storage disease.

Morphological and biochemical studies were performed on the CNS of neurologically affected NCTR-Balb/C mouse. Histological and electron microscopic techniques demonstrated severe myelin deficiency in the affected brains. Neither the presence of lipid-containing macrophages nor reactive gliosis was apparent. Analysis of myelin-associated lipids and proteins revealed prominent depletion of galactocerebroside, sulfatide, and proteolipid proteins. In contrast to the scarcity of myelin-specific constituents a marked accumulation of GM2 and GM3 gangliosides and several neutral glycolipids, i.e., glucocerebroside, lactosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide were found in affected CNS. These abnormalities were already apparent in 12-day-old pups as well as in 65-day-old mice. A significant deficit in the proportion of long-chain fatty acids (C24), notable in both normal and alpha-hydroxy acids of cerebrosides from affected white matter, was measured. The lack of reactive gliosis, the observed depletion of galactocerebroside and sulfatide at the early age of 12 days, and the relative decrease in long-chain fatty acids in affected CNS strongly suggest a defect in myelinogenesis in this mutant rather than a secondary process of myelin breakdown.

Animals↗
PubMed1984

Differential salt fractionation of active and inactive genomic domains in chicken erythrocyte.

We have utilized the Sanders salt fractionation technique (Sanders, M. M. (1978) J. Cell Biol. 79, 97-109) to analyze the products of micrococcal nuclease digestion of adult chicken erythrocyte nuclei. By dot-blot hybridization with specific gene probes, it is found that nucleosomes from the globin gene domain, including a region extending to about 10 kilobase pairs 5' to the beta p gene are selectively enriched in the fractions eluted at low salt. In contrast, a single copy sequence located at about 10 kilobase pairs 5' to the beta p gene was concentrated in the less salt-soluble fractions. The vitellogenin and ovalbumin genes, which are never expressed in erythroid tissues, are also concentrated in the less salt-soluble fractions. Some more generally expressed genes (histone H4, thymidine kinase) appear to be more uniformly distributed. The low salt fractions are depleted in H1/H5, enriched in high mobility group 14 and 17, and contain somewhat more highly acetylated histones.

Animals↗
PubMed1984

Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis.

As an alternative approach to classical genetic analysis, we are investigating the potential of anti-sense (nonsense) DNA strand transcription to inhibit gene activity. A promoter will direct transcription of the complementary nonsense DNA strand when the protein coding sequence of a cloned gene is excised and then reinserted in reverse orientation. When such flipped gene constructions of the HSV thymidine kinase (TK) gene are coinjected with the wild-type gene at a 100:1 ratio, there is a reduction of transient TK expression in TK- mouse L cells. The proportion of viable cells with demonstrable TK activity drops 4-fold as compared with neighboring cells coinjected with TK and an excess of control plasmid. Furthermore, autoradiography of the cells still expressing TK shows that 3H-thymidine incorporation is reduced. Cells contransformed with flipped TK gene constructions have a reduced capacity to express subsequently microinjected TK genes, suggesting that the anti-message phenomenon is due to a trans-inhibition of TK and is probably not an artifact of rearrangements following microinjection.

Animals↗
PubMed1984

Histone-H1-dependent chromatin superstructures and the suppression of gene activity.

I have identified a chromatin particle containing DNA as large as 20-40 kb that migrates as a discrete entity on agarose gels. With increasing nuclease digestion, the particle becomes cleaved in the linker regions between nucleosomes, but remains intact, probably held together by the outer histones, H1 and H5. By hybridization analysis, inactive genes are found in these particles. Active genes (and their flanking sequences) are also found in particles containing H1 and H5, but in contrast to inactive supranucleosome particles, active polynucleosome particles are not held together after cleavage of linker DNA. This suggests that H1 cross-links adjacent nucleosomes in inactive regions and that H1 is bound differently in expressed regions. The results raise the possibility that the marked degree of suppression of repressed, tissue-specific genes may be determined, in part, by their assembly into these inactive supranucleosome structures.

Animals↗
PubMed1984

Multiple tropomyosin polypeptides in chicken embryo fibroblasts: differential repression of transcription by Rous sarcoma virus transformation.

We have found that cytoskeletal extracts of cultured chicken embryo fibroblasts contain at least seven distinct polypeptides (two major and five minor) which cross-react with antiserum to chicken smooth muscle tropomyosin. These polypeptides range in apparent molecular weight from 31,000 to 47,000, and each is encoded by mRNAs which specifically hybridize to cloned muscle tropomyosin cDNAs. These nonmuscle tropomyosin species and their respective mRNAs are electrophoretically distinct from those of chicken skeletal muscle and appear by genomic DNA blotting to comprise a part of a multigene tropomyosin family. In Rous sarcoma virus-transformed chicken embryo fibroblasts, synthesis of the tropomyosins is differentially repressed such that the synthesis of the major species (cp35 and cp33, cytoskeletal proteins of molecular weight 35,000 and 33,000, respectively) and three minor species is drastically reduced, whereas the synthesis of two of the minor species (cp32 and cp31) remains essentially unchanged. Analysis of cellular mRNA and runoff nuclear transcription experiments indicate that the repression of tropomyosin synthesis by Rous sarcoma virus transformation occurs at the level of transcription. This repression of tropomyosin synthesis is partially mimicked in normal chicken embryo fibroblasts during incubation in high-NaCl medium, a condition in which chicken embryo fibroblasts acquire many characteristics of transformed cells.

Animals↗
PubMed1984

Differences in lipid composition of doxorubicin-sensitive and -resistant P388 cells.

An anthracycline-resistant subline of P388 murine leukemia cells, P388/ADR, has been shown to have an altered total lipid content and composition when compared to the parent line. When specific lipids of the cell lines are compared, it is found that P388/ADR cells contain approximately 3.6 times the amount of triglycerides as P388 cells. Although no differences are noted in the amounts of unesterified cholesterol or total phospholipids in the two cell lines, they do differ in specific phospholipid patterns. P388/ADR cells contain relatively less phosphatidylcholine and more sphingomyelin than drug-sensitive cells. No differences are observed in the content of phosphatidylethanolamine and cardiolipin. The difference in phosphatidylcholine/sphingomyelin ratio (5.57 for ADR-sensitive cells and 3.28 for ADR-resistant cells) may account for the previously observed difference in plasma membrane lipid structural order between these cell lines. Measurements of the relative activity of phosphocholine transferase in the two lines, using the rate of incorporation of 3H-choline into phosphatidylcholine, indicate that lower phosphocholine transferase activity in P388/ADR cells may account for the observed changes in cellular lipid and phospholipid composition.

Animals↗
PubMed1983

Transcription of DNA injected into Xenopus oocytes is influenced by template topology.

We have assayed the transcription of the herpes simplex virus thymidine kinase gene on circular and linear plasmids after injection into Xenopus oocytes. The circular plasmids appear to be transcribed at least 500 times more efficiently than the linear plasmids. Moreover, when the circular templates are allowed to form functioning transcription complexes, and are then linearized in situ, the synthesis of accurate transcripts is substantially reduced.

Animals↗
PubMed1983

A dominant role for DNA secondary structure in forming hypersensitive structures in chromatin.

Previous work has suggested that potential information in DNA secondary structure might be used by cells to define DNAase 1- and S1-sensitive chromatin structures associated with promoter and terminator regions. To test this hypothesis, supercoiled pBR322 was cotransfected into L cells. For the majority of transfected clones supercoil-induced S1-sensitive sites in pure pBR322 DNA are also S1-sensitive in L-cell nuclei. These results suggest that the potential of certain DNA sequences to form specific secondary structures in chromatin can be a dominant characteristic. A recombinant chicken beta A-globin supercoiled plasmid was reconstituted in vitro with histones. The reconstituted chromatin also retained the ability to form S1-sensitive sites. Evidence suggests that DNA sequences capable of forming S1-sensitive sites in supercoiled plasmids may bind nucleosomes poorly after reconstitution with histones.

Animals↗
PubMed1983

Temporal order of chromatin structural changes associated with activation of the major chicken vitellogenin gene.

The major chicken vitellogenin (VTG II) gene is marked at the chromatin level in hormone-responsive cells by a set of nuclease-hypersensitive sites that exist prior to hormone-mediated expression. When the gene is transcriptionally activated in the liver by treatment with 17 beta-estradiol, three additional hypersensitive sites are induced, which map near the 5' end of the gene. Two of these sites are stable and, moreover, appear to be propagated to daughter cells after hormone is withdrawn. In contrast, the third site (located 0.7 kb upstream from the gene) is observed only during periods of hormone treatment, perhaps due to a transient interaction with hormone receptor complex at this site. Whereas none of these nuclease-hypersensitive sites is observed in erythrocytes, brain, or fibroblasts, a subset is present in differentiated oviduct cells that have estrogen receptors, but do not express VTG II.

Animals↗
PubMed1983

Detection of an altered DNA conformation at specific sites in chromatin and supercoiled DNA.

We show that bromoacetaldehyde, which reacts selectively at the N-1 and N-6 positions of unpaired adenine and at the N-3 and N-4 positions of unpaired cytosine residues reacts with chromosomal DNA in intact cells at probable regulatory sequences near active genes. A region of about 200 base pairs 5' to the chicken beta A-globin gene, which contains sites sensitive to nuclease S1, to several restriction endonucleases, and to very low levels of DNase I, also contains DNA structures that are preferentially sensitive to bromoacetaldehyde. These altered DNA structures are found at reproducible positions relative to the beta A-globin gene regardless of whether the bromoacetaldehyde is presented to intact erythrocytes, erythrocyte nuclei, or the beta A-globin gene itself carried in pBR322 as purified supercoiled DNA. The unpaired DNA 5' to the adult beta A-globin gene in adult erythrocytes is not detectable in embryonic erythrocytes that express embryonic beta-globin in contrast to adult beta A-globin. Our results suggest that well-defined regions of DNA with effectively unpaired bases occur in intact nuclei and that these structures may be important for specific recognition because they are tissue specific and are found at putative regulatory regions.

Acetaldehyde↗